EP1112352A2 - Methyltransferases, nucleic acid molecules encoding methyltransferases, their recombinant expression and uses thereof - Google Patents

Methyltransferases, nucleic acid molecules encoding methyltransferases, their recombinant expression and uses thereof

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Publication number
EP1112352A2
EP1112352A2 EP99944425A EP99944425A EP1112352A2 EP 1112352 A2 EP1112352 A2 EP 1112352A2 EP 99944425 A EP99944425 A EP 99944425A EP 99944425 A EP99944425 A EP 99944425A EP 1112352 A2 EP1112352 A2 EP 1112352A2
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Prior art keywords
nucleotide
seq
εequence
dna
sequence
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German (de)
French (fr)
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Tapani Reinikainen
Antti NYYSSÖLÄ
Janne Kerovuo
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Danisco Finland Oy
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Danisco Finland Oy
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)

Definitions

  • Methyltransferases nucleic acid molecules encoding methyltransferases, their recombinant expression and uses thereof
  • the present invention relates to proteins which are capable of functioning as methyltransferases. More, specifically, the present invention relates to methyltransferases which are capable of carrying out at least one of the following reactions : the conversion of glycine to sarcosine (N- methylglycine) , sarcosine to N,N-dimethyl glycine and N,N- dimethyl glycine to betaine (N,N,N-trimethylglycine) in the presence of a methyl group donor. Furthermore, the present invention relates to nucleic acid molecules encoding such methyltransferase proteins, recombinant organisms which are capable of expressing said nucleic acids as well as the use of said recombinant organisms .
  • Betaine N,N,N-trimethylglycine
  • Betaine is synthesized or accumulated in living cells in response to abiotic stress (salinity, desiccation or low temperatures) (Mc Gue and Hanson, 1990; Csonka, 1989; Yancey et al . , 1982; Wyn Jones et al . , 1977; Gorham, 1995; Bohnert and Jensen, 1996) . Due to its physical properties, betaine is an osmolyte and thus it is able to restore and maintain osmotic balance of living cells. In addition, it has been demonstrated that betaine stabilizes and protects cell membranes (Coughlan and Heber, 1982) and other macromolecules (i.e. enzymes) in the cell (Papagiorgiou and Murata, 1995) .
  • Betaine is synthesized by a number of microbes.
  • these microbes are usually capable of accumulating betaine or a precursor, choline, from the culture medium (Boch et al., 1994; Perroud and Rudulier, 1985; Kempf and Bremer, 1995; Glaasker et al., 1996; Peter et al . , 1996; and Kappes et al., 1996) .
  • Practically all halophilic bacteria are able to use betaine as at least one of their osmolytes in order to survive in the high ionic strength environment .
  • betaine In animal cells, betaine also acts as a methyl group donor. The most important function is to the ability to methylate homocysteine back to methionine, which can then further be used as a methyl group donor when metabolized to S-adenosyl methionine (SAM) . It has been demonstrated that orally administered betaine relieves diarrhea and dehydration in many animals and inhibits invasion of gut epithelium by coccidia parasite (Ferket, 1994; Augustine and McNaughton, 1996) . As a methyl group donor betaine has been shown to be lipotropic, thus decreasing the amount of fat in chicken meat (Saunderson and MacKinlay, 1990; Barak et al . , 1993) .
  • the most extensively studied betaine biosynthesis pathway is the two-step oxidation reaction of choline to betaine via betaine aldehyde. This metabolic pathway has been demonstrated to exist in number of microbes, plants and animal cells.
  • the choline-betaine pathway of E. coli (La ark et al., 1991) and Pseudomonas aeruginosa (Nagasawa et al . , 1976) comprises an oxygen-dependent choline dehydrogenase, which catalyzes the oxidation of both choline to betaine aldehyde and betaine aldehyde to betaine.
  • the E. coli choline dehydrogenase gene has been cloned and sequenced.
  • oxidation of choline to betaine can be catalyzed by a choline oxidase found for example in some Corynebacteria, Brevibacterium and Alcaligenes species
  • the second oxidation step from betaine aldehyde to betaine may also be catalyzed by a betaine aldehyde dehydrogenase.
  • a betaine aldehyde dehydrogenase has also been found in a number of organisms (Pseudomonas aeruginosa (30) ) . Also some plants have this enzyme (Hanson et al., 1985) . In plants, the enzyme has been demonstrated to have wider substrate specificity and thus it also catalyzes other reactions (Trossat et al . , 1997) . The gene has been cloned from E.
  • E. coli (Lamark et al., 1991), spinach (Weretilnyk and Hanson, 1990) and also from barley (Ishitani et al . , 1995) .
  • the E. coli gene has also successfully been expressed in transgenic tobacco (Holmstr ⁇ m et al . , 1994).
  • Betaine is used as feed additive in feed industry.
  • transgenic plants producing high amounts of betaine in vivo would have better nutritional value.
  • Feed crops e.g. maize or soybean
  • betaine could therefore directly be used in feed without the need of betaine supplementation.
  • betaine is synthesized by many plants, there are several commercially important crops such as potato, rice, tomato and tobacco which do not accumulate betaine.
  • Bulow and co-workers (1995) were the first to demonstrate that expression of the E. coli choline dehydrogenase in tobacco improves the salt tolerance and freezing tolerance (Holmberg, 1996) of transgenic potato and tobacco due to endogenously synthesized betaine.
  • Arabidopsis Hidet al., 1997)
  • rice Neakamura et at., 1997; Guo, 1997) . Therefore, expression of the methyltransferases in plants can facilitate stress tolerance and improve the productivity of the plants when grown under conditions of water stress or freezing and cold temperatures.
  • betaine has shown to induce pathogenesis-related protein expression in plants (Xin et al . , 1996) as well as increasing the resistance of plants to attack by pathogenic fungi or nematodes (Blunden et al . , 1996; Wu et al . ,1997) and may decrease the incidence of nematode (e.g. Meloidogne javanica and M. incognita) attack in plants. Therefore, transgenic plants producing endogenous betaine, can be more resistant to fungal pathogens. Moreover, the betaine synthesis in the plants may be coupled to the systemic resistance genes which are induced when the plant is attacked by pathogens .
  • Endogenously synthesized betaine may also affect the viability of microbes and therefore it would improve their performance in various biotechnical processes. For instance, in high cell density fermentation or immobilized cell systems, the production microbes are subjected to considerable environmental stress. Betaine has successfully been used in fermentation media to increase the product yield in amino acid production. For instance, betaine has shown to relieve stress and improve yield of lysine producing Brevibacterium lactofermentum (Kawahara et al . , 1990). Betaine is also commercially sold for the purpose (Nutristim®, Cultor Corp) . Thus, endogenously synthesized betaine can improve productivity in biotechnical processes where the cells are subjected to abiotic stress.
  • Microbes also suffer from stress when subjected to high temperatures or when cells are freeze-dried or frozen.
  • the viability of yeast or bacterial cells may be dramatically reduced in these processes used in e.g. frozen dough manufacturing or preservation of lactic acid bacterium starters. Therefore, it would be highly advantageous if one could improve the viability of microbes subjected to freeze-thaw or freeze-drying processes by accumulating betaine inside the cells.
  • exogenously applied betaine improves the viability of microbes in extreme pH (Smirnova and Oktyabrsky, 1995; Chambers and Kunin, 1985) .
  • Improved performance of beneficial, probiotic microbes organisms in animal digestive tract can be utilized in animal nutrition.
  • introduction of a betaine synthesis pathway can improve the stress tolerance of "probiotic" lactic acid microbes which efficiently bind to gut epithelium in cells, providing a way to balance the microbial population in the GI tract and to improve pathogen resistance.
  • Betaine has been shown to stabilize proteins in the cells .
  • cytoplasmic accumulation of betaine will reduce the formation of inclusion bodies (Blackwell and Horgan, 1991; Bhandari and Gowrishankar, 1997) which is a problem often encountered when heterologous proteins are expressed in E. coli.
  • co-expression of the genes of betaine biosynthesis with the protein of interest should result in better solubility of the heterologous protein and reduce the amount of inclusion bodies .
  • the use of the glycine methylation pathway may have a number of advantages over the oxidative synthesis from choline.
  • Glycine is synthesized by practically all organisms and as an amino acid, this metabolite is present in high concentrations in the cells.
  • the availability of intracellular choline may limit betaine biosynthesis.
  • the metabolism and formation of glycine in cells is known and the genes of this metabolic pathway have been cloned, thus allowing the engineering of the glycine pathway.
  • an object of the present invention is to provide proteins which are capable of acting in a biosynthetic pathway from glycine to betaine as well as methods for the purification and production of said proteins.
  • a further object of the present invention is to provide nucleic acid molecules which, when transformed into a host organism, encode proteins which are capable of acting in a biosynthetic pathway from glycine to betaine.
  • a further object is to provide recombinant microorganisms which are capable of expressing one or more proteins which are capable of acting in a biosynthetic pathway from glycine to betaine for the production of betaine and precursors thereof .
  • an object of the present invention is to provide recombinant plants which express one or more proteins which are capable of acting in a biosynthetic pathway from glycine to betaine for the production of betaine and precursors thereof .
  • an object of the present invention is to provide a method for the production of betaine and precursors thereof, for example sarcosine and dimethyl glycine, in recombinant organisms .
  • a further object is to provide recombinant organisms which have an increased concentration of intracellular betaine and are useful in the fields of recombinant heterologous protein production, agriculture, etc.
  • a further object of the present invention is to provide nucleic acid probes and method for identifying and cloning genes which encode proteins which are capable of participating in a biosynthetic pathway from glycine to betaine.
  • a further object of the present invention is to provide methods for improving the general growth and/or productivity of an organism including enhancing stress tolerance, for example, salt tolerance, freezing tolerance and cold tolerance, enhancing resistance to drought, water stress and attack by pathogens in organisms.
  • stress tolerance for example, salt tolerance, freezing tolerance and cold tolerance
  • an object of the present invention is to provide recombinant microorganisms which have improved viability in culture, enhanced pH tolerance in culture, result in decreased inclusion body formation when expressing a heterologous protein, result in increased solubility, stability and/or yield of a heterologous protein expressed in said organism.
  • the inventors have identified, isolated and purified proteins which are capable of carrying out at least one of the following reactions in a metabolic pathway from glycine to betaine: the conversion of glycine to sarcosine (N- methylglycine) , sarcosine to N,N-dimethyl glycine and N,N- dimethyl glycine to betaine (N,N,N-trimethylglycine) in the presence of a methyl group donor.
  • These proteins are designated herein as methyltransferases based on their ability to transfer a methyl group from a methyl group donor to a methyl group acceptor.
  • the above mentioned reactions are individual steps in a three-step methylation reaction pathway of glycine to betaine in certain microorganisms, for example, Ectothiorhodospira halochloris and Actinopolyspora halophila.
  • a methyltransferase of the present invention (designated hereinafter as glycine-sarcosine methyltransferase or GSMT) has been isolated from Ectothiorhodospira halochloris and Actinopolyspora halophila which is capable of catalyzing methylation reactions that convert glycine to dimethyl glycine, i.e. convert glycine to sarcosine (N-methyl glycine) and sarcosine to dimethyl glycine (N,N-dimethyl glycine) .
  • a methyltransferase according to the present invention (designated hereinafter as sarcosine- dimethylglycine methyltransferase or SDMT) has been isolated from Ectothiorhodospira halochloris and Actinopolyspora halophila which is capable of catalyzing methylation reactions that convert sarcosine to betaine, i.e. convert sarcosine (N-methyl glycine) to dimethyl glycine (N,N- dimethyl glycine) and dimethyl glycine to betaine.
  • GSMT glycine-sarcosine me- methyltransferase lycine
  • the methyltransferases of the present invention are capable of utilizing S-adenosyl methionine (hereinafter also referred to as SAM) as a methyl group donor in the above reactions.
  • SAM S-adenosyl methionine
  • Figure 1 Formation of methylation products from glycine, sarcosine and dimethyl glycine substrates using the A . halophila cell extract. The retention times of the standards are shown by arrows .
  • FIG. 1 Analysis of purified methyl transferases on SDS- PAGE.
  • Figure 3 The determination of the isoelectric point of A . halophila SDMT by isoelectric focusing.
  • FIG. 5 The temperature dependence of A. halophila SDMT activity.
  • Figure 6 In vi tro synthesis of betaine by using the purified E. halochloris GSMT and A. halophila SDMT enzymes. The retention times of the standards are shown by arrows.
  • Figure 7 The schematic structure of the betaine operons of A . halophila and E. halochloris . GSMT; glycine sarcosine methyltransferase. SDMT; sarcosine dimethyl glycine methyltransferase. SAMS; S-adenosyl methionine synthase.
  • Figure 8 The nucleotide and amino acid sequence of the E. halochloris betaine operon.
  • the arrows indicate the amino acids encoding GSMT, SDMT and SAMS.
  • the underlined regions indicate regions which are hybridized with the primers used to construct the expression vectors in heterologous organisms.
  • the * indicates a stop codon.
  • Figure 9 The nucleotide and amino acid sequence of the A . halophila betaine operon.
  • the underlined regions indicate regions which are hybridized with the primers used to construct the expression vectors in heterologous organisms.
  • the * indicates a stop codon.
  • Figure 10 Schematic presentation of the expression plasmid used in expression of the methyl transferases.
  • the insert was ligated to vector digested with Ncol/Bgrlll .
  • FIG. 11 The growth curves of E. coli transformants carrying the E. halochloris GSMT gene (EGSM) .
  • Transformant carrying only the cloning vector (PQE-60) was used as the control .
  • FIG. 12 The growth curves of E. coli transformants carrying the E. halochloris GSMT and SDMT genes (EhFU) . Transformant carrying only the cloning vector (PQE-60) was used as the control.
  • a methyltransferase for example GSMT, capable of catalyzing the conversion of glycine to sarcosine ( ⁇ -methyl glycine) and/or the conversion of sarcosine to dimethyl glycine ( ⁇ , ⁇ - dimethyl glycine) .
  • said methyltransferase comprises an amino acid sequence selected from the group consisting of:
  • a fragment of an amino acid sequence as depicted in SEQ ID NO: 2 or SEQ ID NO : 6 is designated as any fragment of an amino acid sequence as depicted in SEQ ID NO: 2 or SEQ ID NO: 6 which is capable of catalyzing the conversion of glycine to sarcosine (N-methyl glycine) and/or the conversion of sarcosine to dimethyl glycine (N,N-dimethyl glycine) .
  • a derivative of an amino acid sequence as depicted in SEQ ID NO: 2 or SEQ ID NO: 6 is designated as any mutation, deletion, addition, substitution, insertion or inversion of one or more amino acids in the of amino acid sequence as depicted in SEQ ID NO: 2 or SEQ ID NO : 6 which is capable of catalyzing the conversion of glycine to sarcosine (N-methyl glycine) and/or the conversion of sarcosine to dimethyl glycine (N,N-dimethyl glycine) .
  • a derivative of a methyltransferase which is capable of catalyzing the conversion of glycine to sarcosine (N-methyl glycine) and/or the conversion of sarcosine to dimethyl glycine (N,N-dimethyl glycine) has about 60 % homology, preferably about 70 % homology, more preferably about 80 % homology, and most preferably about 90 % homology to the corresponding amino acid sequence depicted in SEQ ID NO : 2 or SEQ ID NO: 6.
  • the amino acids constituting these changes are selected from the 20 standard naturally-occurring amino acids found in proteins, i.e. Ala, Val, Leu, lie, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, Asp, Glu, Lys , Arg and His.
  • the mutation (s) and/or substitution (s) are conservative, for example, Ala, Val, Leu, lie, Pro, Phe, Trp, or Met residue (s) are replaced with one of these amino acids, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, residue (s) are replaced with one of these amino acids , Asp or Glu are replaced with one of these amino acids and Lys, Arg or His are replaced with one of these amino acids .
  • a methyltransferase according to the invention has the amino acid sequence depicted in SEQ ID NO: 2.
  • a methyltransferase according to the invention has the amino acid sequence depicted in SEQ ID NO: 6.
  • the methyltransferase of the present invention capable of catalyzing the conversion of glycine to sarcosine (N-methyl glycine) and/or the conversion of sarcosine to dimethyl glycine (N,N-dimethyl glycine) can exist in the form of an active enzyme or as a zymogen.
  • the term 'zymogen' designates a protein molecule or fragment or derivative thereof (as defined above) which is synthesized in an inactive form and is capable of being activated in vitro or in vivo by the chemical or enzymatic cleavage of one or more peptide bonds .
  • a preferred zymogen of the methyltransferase of the present invention capable of catalyzing the conversion of glycine to sarcosine (N-methyl glycine) and/or the conversion of sarcosine to dimethyl glycine (N,N-dimethyl glycine) comprises the amino acid sequence as depicted in SEQ ID NO: 2 and SEQ ID NO: 3, wherein the N-terminus of SEQ ID NO: 3 is joined to the C- erminus of SEQ ID NO: 2.
  • a further embodiment of the present invention provides a methyltransferase, for example SDMT, capable of catalyzing the conversion of sarcosine (N-methyl glycine) to dimethyl glycine (N,N-dimethyl glycine) and/or dimethyl glycine to betaine.
  • a methyltransferase for example SDMT, capable of catalyzing the conversion of sarcosine (N-methyl glycine) to dimethyl glycine (N,N-dimethyl glycine) and/or dimethyl glycine to betaine.
  • said methyltransferase comprises an amino acid sequence selected from the group consisting of :
  • a fragment of an amino acid sequence as depicted in SEQ ID NO: 3 or SEQ ID NO: 7 is designated as any fragment of an amino acid sequence as depicted in SEQ ID NO: 3 or SEQ ID NO: 7 which is capable of catalyzing the conversion of sarcosine (N- methyl glycine) to dimethyl glycine and/or the conversion of dimethyl glycine to betaine.
  • a derivative of an amino acid sequence as depicted in SEQ ID NO: 3 or SEQ ID NO: 7 is designated as any mutation, deletion,- addition, substitution, insertion or inversion of one or more amino acids, or combination thereof, in the of amino acid sequence as depicted in SEQ ID NO: 3 or SEQ ID NO: 7 which is capable of catalyzing the conversion of sarcosine (N-methyl glycine) to dimethyl glycine and/or the conversion of dimethyl glycine (N,N-dimethyl glycine) to betaine.
  • a derivative of a methyltransferase which is capable of catalyzing the conversion of sarcosine (N-methyl glycine) to dimethyl glycine and/or the conversion of dimethyl glycine (N,N-dimethyl glycine) to betaine has about 60 % homology, preferably about 70 % homology, more preferably about 80 % homology, and most preferably about 90 % homology to the corresponding amino acid sequence depicted in SEQ ID NO: 3 or SEQ ID NO: 7.
  • amino acids constituting these changes are selected from the 20 standard naturally-occurring amino acids found in proteins, i.e. Ala, Val, Leu, lie, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, Asp, Glu, Lys, Arg and His.
  • the mutation (s) and/or substitution (s) are conservative, for example, Ala, Val, Leu, lie, Pro, Phe, Trp, or Met residue (s) are replaced with one of these amino acids, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, residue (s) are replaced with one of these amino acids, Asp or Glu are replaced with one of these amino acids and Lys, Arg or His are replaced with one of these amino acids .
  • a methyltransferase according to the invention has the amino acid sequence depicted in SEQ ID NO: 3.
  • a methyltransferase according to the invention has the amino acid sequence depicted in SEQ ID NO: 7.
  • the methyltransferase of the present invention capable of catalyzing the conversion of sarcosine (N-methyl glycine) to dimethyl glycine and/or the conversion of dimethyl glycine (N,N-dimethyl glycine) to betaine can exist in the form of an active enzyme or as a zymogen.
  • the term 'zymogen' designates a protein molecule, fragment or derivative thereof (as defined above) which is synthesized in an inactive form and is capable of being activated in vitro or in vivo by the chemical or enzymatic cleavage of one or more peptide bonds .
  • a preferred zymogen of the methyltransferase of the present invention capable of catalyzing the conversion of sarcosine (N-methyl glycine) to dimethyl glycine and/or the conversion of dimethyl glycine (N,N-dimethyl glycine) to betaine comprises the amino acid sequence as depicted in SEQ ID NO: 2 and SEQ ID NO: 3, wherein the N-terminus of SEQ ID NO: 3 is joined to the C-terminus of SEQ ID N0:2.
  • the methyltransferases of the present invention for example naturally occurring GSMT and SDMT or recombinantly produced GSMT and SDMT and fragments or derivatives thereof as well as zymogens of these naturally occurring or recombinant proteins, are preferably isolated to a state free from other proteins originating from the organisms from which they are isolated; more preferably, to a pure state, most preferably to a homogeneous state .
  • SAM S-adenosyl methionine
  • SAMS S-adenosyl methionine synthase
  • said SAMS comprises an amino acid sequence selected from the group consisting of:
  • a fragment of an amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO : 8 is designated as any fragment of an amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO: 8 which is capable of catalyzing the conversion of S-adenosyl methionine from methionine and ATP (adenosine triphosphate) .
  • a derivative of an amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO : 8 is designated as any mutation, deletion, addition, substitution, insertion or inversion of one or more amino acids, or combination thereof, in the of amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO: 8 which is capable of catalyzing the conversion of S-adenosyl methionine from methionine and ATP.
  • a derivative which is capable of catalyzing the conversion of methionine to S- adenosyl methionine has about 60 % homology, preferably about 70 % homology, more preferably about 80 % homology, and most preferably about 90 % homology to the corresponding amino acid sequence depicted in SEQ ID NO: 4 or SEQ ID NO: 8.
  • the amino acids constituting these changes are selected from the 20 standard naturally-occurring amino acids found in proteins, i.e.
  • the mutation (s) and/or substitution (s) are conservative, for example, Ala, Val, Leu, lie, Pro, Phe, Trp, or Met residue (s) are replaced with one of these amino acids, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, residue (s) are replaced with one of these amino acids, Asp or Glu are replaced with one of these amino acids and Lys, Arg or His are replaced with one of these amino acids .
  • the SAMS of the invention has the amino acid sequence depicted in SEQ ID NO: 4.
  • the SAMS of the invention has the amino acid sequence depicted in SEQ ID NO: 8.
  • the SAMS of the present invention for example naturally occurring SAMS or recombinantly produced SAMS and fragments or derivatives thereof, are preferably isolated to a state free from other proteins originating from the organisms from which they are isolated; more preferably, to a pure state, most preferably to a homogeneous state.
  • the present invention also relates to a nucleic acid molecule which is capable of encoding a methyltransferase capable of converting glycine to sarcosine (N-methyl glycine) and/or sarcosine to dimethyl glycine (N,N-dimethyl glycine) .
  • a nucleic acid molecule which encodes a methyltransferase capable of converting glycine to sarcosine (N-methyl glycine) and/or sarcosine to dimethyl glycine (N,N-dimethyl glycine) comprises a nucleotide sequence selected from the group consisting of:
  • a nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO : 1 or a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5 is designated as any nucleotide sequence, for example DNA or RNA, preferably DNA, which hybridizes under standard conditions to a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO: 1 or a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5 and encodes a methyltransferase capable of converting glycine to sarcosine (N-methyl glycine) and/or sarcosine to dimethyl glycine (N,N-dimethyl glycine) .
  • the term 'standard conditions' designates a standard procedure used in heterologous hybridization to screen for genes with enough sequence homology (Maniatis, 1989) .
  • the hybridization filters are washed first in low stringency conditions and the stringency is gradually increased (usually by increasing the washing temperature) in order to select the positive signals from the background.
  • plaques or colonies of a gene bank to be screened can be transferred onto nitrocellulose membranes and hybridized with a PCR fragment, oligonucleotide or any other cloned DNA containing a fragment of the above gene of interest .
  • the probe can be prepared for example by PCR as described in Example 6.
  • Hybridization can be carried out at 42°C in 50 mM Tris-HCl, pH 7.5 , 10 mM EDTA, 1 M NaCI, 0.5% SDS, 0.1% sodium pyrophosphate, lOx Denhardt ' s solution (Maniatis, 1989) , 100 ⁇ g herring sperm DNA and 125 ⁇ g/ml polyA.
  • the filters can be first washed with low stringency conditions, for example, at 37°C using 3x SSC, 0.5% SDS and 10% sodium pyrophosphate (Maniatis, 1989) . The filters can then be exposed to x-ray film to monitor the number of positive clones .
  • the washing temperature will be raised in 2-5°C intervals up to a temperature at which the background becomes invisible and only a few positive plaques are obtained.
  • the positive plaques or colonies can then be purified and the DNA can be isolated for Southern blot hybridization to check the size of the cloned insert for example.
  • the cloned DNA obtained can be sequenced. On the basis of the sequence homology, it can be concluded that the DNA contains the gene of interest .
  • a nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO: 1 or a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5 has about 60 %, preferably 70 %, more preferably 80 % and especially 90 % homology to a nucleotide sequence corresponding to a DNA sequence comprising nucleotide 208 to 1047 of SEQ ID NO:l or a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5.
  • a fragment of a nucleotide sequence from nucleotide 208 to 1047 of SEQ ID NO : 1 or a nucleotide sequence from nucleotide 221 to 1024 of SEQ ID NO : 5 is designated as any nucleic acid fragment, for example DNA or RNA, preferably DNA, which encodes a methyltransferase capable of converting glycine to sarcosine (N-methyl glycine) and/or sarcosine to dimethyl glycine (N,N-dimethyl glycine) .
  • a preferred nucleic acid molecule according to the invention comprises the DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l.
  • a further preferred nucleic acid molecule according to the invention comprises the DNA sequence from nucleotide 221 to 1024 of SEQ ID NO : 5
  • nucleic acid molecules according to the invention comprise the DNA sequence from nucleotide 221 to 1867 of SEQ ID NO : 5 or the DNA sequence from nucleotide 208 to 1902 of SEQ ID NO:l, the DNA sequence from nucleotide 221 to 1867 of SEQ ID NO : 5 being more preferred.
  • the present invention also relates to a nucleic acid molecule which encodes a methyltransferase capable of converting sarcosine (N-methyl glycine) to dimethyl glycine and/or dimethyl glycine (N,N-dimethyl glycine) to betaine.
  • a nucleic acid molecule which encodes a methyltransferase capable of converting sarcosine (N-methyl glycine) to dimethyl glycine and/or dimethyl glycine (N,N-dimethyl glycine) to betaine comprises a nucleotide sequence selected from the group consisting of:
  • a nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO: 1 or a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO : 5 is designated as any nucleotide sequence, for example DNA or RNA, preferably DNA, which hybridizes under standard conditions to a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO : 1 or a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO : 5 and encodes a methyltransferase capable of converting sarcosine (N-methyl glycine) to dimethyl glycine and/or dimethyl glycine (N,N-dimethyl glycine) to betaine.
  • any nucleotide sequence for example DNA or RNA, preferably DNA, which hybridizes under standard conditions to a DNA sequence from nucleotide 1048 to 1902 of S
  • the term 'standard conditions' designates a standard procedure used in heterologous hybridization to screen for genes with enough sequence homology (Maniatis, 1989). Basically, the hybridization filters are washed first in low stringency conditions and the stringency is gradually increased (usually by increasing the washing temperature) in order to select the positive signals from the background. For example, plaques or colonies of a gene bank to be screened can be transferred onto nitrocellulose membranes and hybridized with a PCR fragment, oligonucleotide or any other cloned DNA containing a fragment, of the above gene of interest .
  • the probe can be prepared for example by PCR as described in Example 6.
  • Hybridization can be carried out at 42°C in 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1 M NaCI, 0.5% SDS, 0.1% sodium pyrophosphate, lOx Denhardt ' s solution (Maniatis, 1989) , 100 ⁇ g herring sperm DNA and 125 ⁇ g/ml polyA.
  • the filters can be first washed with low stringency conditions, for example, at 37°C using 3x SSC, 0.5% SDS and 10% sodium pyrophosphate (Maniatis, 1989). The filters can then be exposed to x-ray film to monitor the number of positive clones .
  • the washing temperature will be raised in 2-5°C intervals up to a temperature at which the background becomes invisible and only a few positive plaques are obtained.
  • the positive plaques or colonies can then be purified and the DNA can be isolated for Southern blot hybridization to check the size of the cloned insert for example.
  • the cloned DNA obtained can be sequenced. On the basis of the sequence homology, it can be concluded that the DNA contains the gene of interest .
  • a nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 1048 to 1902 of SEQ ID N0:1 or a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5 has about 60 %, preferably 70 %, more preferably 80 % and especially 90 % homology to a nucleotide sequence corresponding to a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l or a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5.
  • a fragment of a nucleotide sequence from nucleotide 1048 to 1902 of SEQ ID NO : 1 or a nucleotide sequence from nucleotide 1031 to 1867 of SEQ ID NO : 5 is designated as any nucleic acid fragment, for example DNA or RNA, preferably DNA, which encodes a methyltransferase capable of converting sarcosine (N-methyl glycine) to dimethyl glycine and/or dimethyl glycine (N,N-dimethyl glycine to glycine betaine.
  • a preferred nucleic acid molecule according to the invention comprises the DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l.
  • a further preferred nucleic acid molecule according to the invention comprises the DNA sequence from 1031 to 1867 of SEQ ID NO:5.
  • nucleic acid molecules according to the invention comprise the DNA sequence from nucleotide 221 to 1867 of SEQ ID NO : 5 or the DNA sequence from nucleotide 208 to 1902 of SEQ ID NO:l, the DNA sequence from nucleotide 221 to 1867 of SEQ ID NO: 5 being more preferred.
  • a further aspect of the present invention provides a nucleic acid molecule which encodes an enzyme capable of converting S-adenosyl methionine from methionine and ATP.
  • a nucleic acid molecule which encodes an enzyme capable of converting S-adenosyl methionine from methionine and ATP comprises a nucleotide sequence selected from the group consisting of:
  • a nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO : 1 or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO : 5 is designated as any nucleotide sequence, for example DNA or RNA, preferably DNA, which hybridizes under standard conditions to a DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO : 1 or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO : 5 and encodes enzyme capable of converting S-adenosyl methionine from methionine and ATP.
  • the term ' standard conditions ' designates a standard procedure used in heterologous hybridization to screen for genes with enough sequence homology (Maniatis, 1989) .
  • the hybridization filters are washed first in low stringency conditions and the stringency is gradually increased (usually by increasing the washing temperature) in order to select the positive signals from the background.
  • plaques or colonies of a gene bank to be screened can be transferred onto nitrocellulose membranes and hybridized with a PCR fragment, oligonucleotide or any other cloned DNA containing a fragment of the above gene of interest .
  • the probe can be prepared for example by PCR as described in Example 6.
  • Hybridization can be carried out at 42 °C in 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1 M NaCI, 0.5% SDS, 0.1% sodium pyrophosphate, lOx Denhardt ' s solution (Maniatis, 1989), 100 ⁇ g herring sperm DNA and 125 ⁇ g/ml polyA.
  • the filters can be first washed with low stringency conditions, for example, at 37°C using 3x SSC, 0.5% SDS and 10% sodium pyrophosphate (Maniatis, 1989) . The filters can then be exposed to x-ray film to monitor the number of positive clones.
  • the washing temperature will be raised in 2-5°C intervals up to a temperature at which the background becomes invisible and only a few positive plaques are obtained.
  • the positive plaques or colonies can then be purified and the DNA can be isolated for Southern blot hybridization to check the size of the cloned insert for example .
  • the cloned DNA obtained can be sequenced. On the basis of the sequence homology, it can be concluded that the DNA contains the gene of interest .
  • a nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 2027 to 2722 of SEQ ID N0:1 or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO: 5 has about 60 %, preferably 70 %, more preferably 80 % and especially 90 % homology to a nucleotide sequence corresponding to a DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO:l or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO: 5.
  • a fragment of a nucleotide sequence from nucleotide 2027 to 2722 of SEQ ID NO : 1 or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO : 5 is designated as any nucleic acid fragment, for example DNA or RNA, preferably DNA, which encodes enzyme capable of converting S-adenosyl methionine from methionine and ATP.
  • a preferred nucleic acid molecule according to the invention comprises the DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO:l.
  • a further preferred nucleic acid molecule according to the invention comprises the DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO : 5.
  • DNA probe for use in identifying and cloning a nucleic acid molecule encoding a methyltransferase comprising at least 15 nucleotide bases, preferably 20 or more nucleotide bases, of a nucleotide sequence selected from the group consisting of : (a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5.
  • Said DNA probes can be utilized in a method according to the invention for identifying and cloning a nucleic acid molecule encoding a methyltransferase comprising the steps of hybridizing said probe with a sample containing nucleic acid of an organism, detecting a nucleic acid molecule in said sample which hybridizes to said probe and isolating said detected nucleic acid molecule.
  • Preferred methods include the use of the polymerase chain reaction (PCR) and Southern blotting techniques which are described herein and are familiar to the skilled person in the art.
  • vectors for expression of the proteins according to the invention in prokaryotic and eukaryotic hosts.
  • expression vectors for example phages, plasmids and DNA or RNA viruses, are capable of transforming and/or replicating and expressing the proteins of the present invention in prokaryotes and/or eukaryotes, for example bacteria, yeast, fungi and/or plants.
  • Such expression vectors and methods for their construction are known to the skilled person and can be provided with nucleic acid elements for transcription, for example start codons, 'TATA' boxes, promoters, enhancers, stop codons, etc., and nucleic acid elements important for translation and processing of the nucleic acids transcribed from said vectors in a given host, for example ribosome binding sites, leader sequences for secretion of the proteins of the present invention, etc.
  • One embodiment of the invention is an expression vector comprising a nucleic acid sequence which encodes a methyltransferase capable of converting glycine to sarcosine (N-methyl glycine) and/or sarcosine to dimethyl glycine (N,N- dimethyl glycine) and/or a nucleic acid sequence which encodes a methyltransferase capable of converting sarcosine (N-methyl glycine) to dimethyl glycine and/or dimethyl glycine (N,N-dimethyl glycine) to betaine.
  • said expression vector comprises at least one nucleotide sequence selected from the group consisting of:
  • an expression vector comprising a nucleotide sequence coding for an enzyme capable of catalyzing the synthesis of S-adenosyl methionine and at least one nucleotide sequence selected from the group consisting of:
  • preferred expression vectors comprise a nucleotide sequence selected from the group consisting of:
  • preferred expression vectors comprise the DNA sequence from nucleotide 208 to 1047 of SEQ ID NO : 1 and the DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO : 1 or the DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5 and the DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5, an expression vector comprising the DNA sequence from nucleotide 221 to 1024 of SEQ ID NO : 5 and the DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO : 5 being more preferred.
  • expression vectors comprising fragments and/or derivatives of the above mentioned sequences as well as other combinations of the above mentioned DNA sequences, for example a DNA sequence from nucleotide 208 to 1047 of SEQ ID N0:1 and a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5 or a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5 and a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l or fragments and/or derivatives thereof are also subject matter of the present invention.
  • expression vectors of the present invention can additionally comprise a nucleic acid molecule coding for an enzyme capable of directly or indirectly increasing the intracellular amount of S-adenosyl methionine.
  • E. halochloris and A. halophila GSMT and SDMT enzymes are located in a "betaine operon".
  • E. halochloris the enzymes are encoded by two separate genes, whereas in A. halophila the two enzymes are coded by a single gene.
  • the "betaine operon” contains a S-adenosyl methionine synthase (SAMS) gene.
  • SAMS enzyme catalyzes the synthesis of S-adenosyl methionine (SAM) from methionine and ATP, and thus, it is useful in the methylation reactions of the methyltransferases of the invention because it increases the concentration of the enzyme substrate SAM. Therefore co-expression of the SAMS gene with one or more of the methyltransferase genes of the invention can be used to increase betaine synthesis in these organisms.
  • the above mentioned expression vectors can additionally comprise a DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO : 1 or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO : 5 or fragments and/or derivatives thereof when the organism to be transformed is E halochloris or A. halophila.
  • Preferred expression vectors of this type which also encode the methyltransferases of the present invention, comprise a DNA sequence from nucleotide 208 to 2722 of SEQ ID NO:l or a DNA sequence from nucleotide 221 to 3004 of SEQ ID NO: 5, the latter being more preferred.
  • said nucleic acid molecule coding for an enzyme capable of directly or indirectly increasing the intracellular amount of S-adenosyl methionine originates or is derived from the organism which is to be transformed with said expression vector.
  • the organism to be transformed with a nucleic acid, for example an expression vector, according to the invention is E. coli
  • nucleic acid for example an expression vector
  • the organism to be transformed with a nucleic acid for example an expression vector
  • a nucleic acid for example an expression vector
  • Other nucleotide sequences which can be used for this purpose are the SAH hydrolase form Mesembryanthemum crystallinum (genebank accession number U79766; Arabidopsis thaliana, accession number AF059581; S. pombe, accession number AL022072) .
  • the expression vectors can additionally comprise a nucleic acid molecule coding for an enzyme capable of increasing the intracellular amount of intracellular glycine.
  • said nucleic acid molecule coding for an enzyme capable of directly or indirectly increasing the intracellular amount of glycine originates or is derived from the organism which is to be transformed with said expression vector.
  • the expression vector can include a nucleotide sequence encoding the enzyme phosphoglycerate dehydrogenase (Bacillus subtilis, accession number L47648; S. pombe, accession number AL022243; Arabidopsis thaliana, accession number AB010407) , phosphoserine aminotransferase (E. coli, accession number AE000193, U00096; Bacillus subtilis, accession number Z99109, AL009126; S. pombe, accession number Z69944; Arabidopsis thaliana, accession number AL031135) , phosphoserine phosphatase (E. coli, accession number AE000509; S.
  • phosphoglycerate dehydrogenase Bacillus subtilis, accession number L47648; S. pombe, accession number AL022243; Arabidopsis thaliana, accession number AB010407
  • Further subject matter of the present invention is a recombinant prokaryotic or eukaryotic organism, for example, bacteria, yeast, fungus or plant, transformed with at least one nucleic acid molecule of the invention as defined above, for example, an expression vector according to the invention as defined above.
  • a recombinant organism according to the invention is a bacterium
  • said bacterium is preferably selected from the group consisting of E. coli, Bacillus, Corynebacteria, Pseudomonas and lactic acid bacteria and Streptomyces .
  • yeast is preferably selected from the group consisting of Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida and Hansenula.
  • the recombinant organism according to the invention is a fungus
  • said fungus is preferably selected from the group consisting of Aspergillus, Trichoderma and Penicillium.
  • the recombinant organism according to the invention is a plant including but not limited to cereals, legumes, oilseeds, vegetables, fruits, ornamentals and perennials
  • said plant is preferably selected from the group consisting of lettuces, Capsicums, grasses, clovers, alfalfa, beans, sweet potatoes, cassava, yams, taro, groundnut, brassica, sugar beet, grapes, potato, tomato, rice, tobacco, rapeseed, maize, sorghum, cotton, soybean, barley, wheat, rye, canola, sunflower, linseed, pea, cucumber, carrot, ornamentals, perennial trees including citrus pear and almond and fruits including strawberry.
  • plant' according to the invention is understood to include individual cells of a plant, plant seeds and callus material.
  • Further subject matter of the present invention is a method for the production of a recombinant organism according to the invention comprising the steps of transforming a host prokaryotic or eukaryotic organism, preferably a bacteria, yeast or fungus, with at least one nucleic acid molecule of the invention as defined above, for example an expression vector according to the invention as defined above.
  • a host prokaryotic or eukaryotic organism preferably a bacteria, yeast or fungus
  • this nucleic acid for example an expression vector
  • this nucleic acid for example an expression vector
  • transformation can be performed using these two nucleic acid molecules, e.g. expression vectors.
  • the present invention relates to a methyltransferase obtainable by culturing wild- ype Ectothiorhodospira or Actinopolyspora or a recombinant prokaryotic or eukaryotic organism according to the invention and isolating said methyltransferase from the organism and/or the medium used to culture or process said organism as well as a method for the production of said methyltransferase comprising the above mentioned steps .
  • a method for the purification of a methyltransferase capable of catalyzing the conversion of glycine to dimethyl glycine comprising the steps of subjecting a sample comprising the methyltransferase to a matrix containing adenosine, binding said methyltransferase to said matrix and eluting said methyltransferase from said matrix is also subject matter of the present invention.
  • the above purification step can be combined with other methods of protein purification including ammonium sulfate precipitation, size exclusion chromatography, cation or anion exchange chromatography, hydrophobic interaction chromatography, etc.
  • Suitable host organisms are practically all bacteria which can be transformed with foreign DNA (for instance E. coli, Bacillus, Corynebacteria, Pseudomonas, lactic acid bacteria and Streptomyces) yeast (for instance Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida and Hansenula) fungi (for instance Trichoderma, Aspergillus, Penicillium) or plants.
  • foreign DNA for instance E. coli, Bacillus, Corynebacteria, Pseudomonas, lactic acid bacteria and Streptomyces
  • yeast for instance Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida and Hansenula
  • fungi for instance Trichoderma, Aspergillus, Penicillium
  • subject matter of the present invention is a method for the production of betaine comprising the steps of culturing a recombinant organism according to the invention and isolating betaine from the organism and/or the medium used to culture or process said organism.
  • sarcosine and/or dimethyl glycine comprising the steps of culturing an organism transformed with a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
  • nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a) , (b) and (c) , and isolating sarcosine and/or dimethyl glycine from said organism or the medium used to culture or process said organism.
  • Further subject matter of the invention is a method for increasing the intracellular concentration of sarcosine, dimethyl glycine and/or betaine in an organism, enhancing the general productivity of an organism, enhancing the salt tolerance of an organism, enhancing the freezing or cold tolerance of an organism, and/or enhancing the resistance of an organism to drought and/or low water stress comprising the steps of transforming an organism with at least one nucleic acid molecule of the invention as defined above, for example an expression vector according to the invention as defined above.
  • said organism is a bacteria, yeast, fungus or plant as recited above .
  • Further subject matter of the invention is a method for inducing pathogenesis-related proteins in a plant, increasing the resistance of a plant to attack by pathogens and/or increasing the nutritional value of a plant comprising the steps of transforming a plant with at least one nucleic acid molecule of the invention as defined above, for example an expression vector according to the invention as defined above.
  • said plant is a plant as recited above.
  • Pathogens include but are not limited to Fusarium sp. which cases root, shoot and leaf diseases in several plant types, Rhizoctonia ⁇ p. and Pythium sp. which cause soil borne diseases in crops, Erysiphe sp. which cause mildew in several species, Phytophthora infestans which causes late blight in potato and tomato, Alternaria solani which causes early blight in potato, fungal diseases of soya caused by Cephalosporium sp., Diaporthe sp., Cerospora sp. Septoria ⁇ p. and Peronospora sp., nematodes for example Meloidogne javanica and M. incognita and insects.
  • subject matter of the invention is a method for enhancing the pH tolerance and/or viability of a cultured microorganism comprising the steps of transforming a microorganism with at least one nucleic acid molecule of the invention as defined above, for example an expression vector according to the invention as defined above.
  • said microorganism is a bacteria, yeast or fungus as recited above .
  • microorganisms of the invention can also be used as hosts in the field of recombinant DNA technology for the expression of a heterologous protein of interest. Therefore, subject matter of the invention is a method for decreasing inclusion body formation, increasing the stability of a heterologous protein and/or increasing the production of a heterologous protein expressed in a microorganism comprising the step ⁇ of transforming a microorganism with at least one nucleic acid molecule of the invention as defined above, for example an expres ⁇ ion vector according to the invention a ⁇ defined above, and tran ⁇ forming a microorganism with a nucleic acid molecule capable of expres ⁇ ing ⁇ aid heterologou ⁇ protein. Said microorganism can be transformed with the nucleic acid molecule, for example an expression vector, according to the invention before, during or after the microorganism is transformed with a nucleic acid molecule capable of expressing said heterologous protein.
  • the growth medium of Actinopolyspora halophila ATCC 27976 used in all cultivations was the "complex medium" described by Sehgal and Gibbons (1960) . Inoculum was grown at 37°C in a shake flask with agitation at 180 rpm until the late exponential growth phase. Then, 8 1 of the above medium with 10 g/l glucose was inoculated with 800 ml culture.
  • the pH in the fermentor (Biostat M (Braun) laboratory fermentor) wa ⁇ maintained at pH 6.5-7.5 with 0.5 M H 2 S0 4 and 1 M NaOH. Agitation and aeration rates were 400 rpm and 10 1/min., respectively.
  • the cultivation temperature was 37°C.
  • Cells were grown to late exponential phase and harve ⁇ ted by centrifugation at 15,000 g for 15 min. Cell ⁇ were stored at -75°C. Before disruption, the cells were thawed and suspended in Buffer I (22 % (w/v) sucrose, 27 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF) , 50 mM Tris-HCl, pH 7.5) in a ratio of 1.5 ml buffer 1:1 g cells (wet weight) .
  • Buffer I 22 % (w/v) sucrose, 27 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF) , 50 mM Tris-HCl, pH 7.5
  • the growth medium used in the cultivation of Ectothiorhodo ⁇ pira halochlori ⁇ ATCC 35916 is described by Tschichholz and Tr ⁇ per (1990) . Cultivation was carried out anaerobically at 42°C in 1 1 glas ⁇ bottle ⁇ with continuou ⁇ ⁇ tirring with magnetic stirrer. The cell ⁇ were illuminated during growth (5,000 - 10,000 lux). 100 ml of pre-inoculum wa ⁇ inoculated into 1 1 medium. Cell ⁇ were grown until late exponential pha ⁇ e and harve ⁇ ted by centrifugation at 28,000 g for 20 min.
  • Buffer II (560 mM Tris-HCl, pH 7.5, 4 mM 2-mercaptoethanol, 50 ⁇ M MgCl2, 160 ⁇ M EDTA) and disrupted with 1 mM PMSF and 1 mM dithiothreitol (DTT) .
  • Buffer II was added in a ratio of 1.5 ml buffer II: 1 g cells (wet weight) .
  • the cells were disrupted with a MSE Soniprep 150 sonicator.
  • the suspension of A. halophila cells wa ⁇ ⁇ onicated in 20 ml batches (sonication pulses 30 s, cooling intervals 2 min) for 1 min / 2 ml cell suspension.
  • the suspension of E. halochloris cells was sonicated in 5 ml batches (sonication pulse ⁇ 15 s; cooling intervals 2 min) for 1 min/1.5 ml cell su ⁇ pension.
  • the cell debris was removed by centrifugation at 28,000 g at 1°C for 30 min.
  • the cell free extracts were stored at -75°C.
  • reaction mixture contained 25 ⁇ l of 0.1 M substrate (glycine, sarcosine or dimethylglycine) , 25 ⁇ l of Buffer II (see above) , 25 ⁇ l 4 mM S-adenosyl-L-methionine containing 45 nCi S-adenosyl-L- [methyl- 14 C] methionine (Amersham) in 1/10 Mcllvaine buffer (pH 3.0), and 25 ⁇ l enzyme sample (e.g. cell free extract) . The reaction was initiated by adding the enzyme. The reaction mixture was incubated for 30 min.
  • substrate glycine, sarcosine or dimethylglycine
  • Buffer II see above
  • 25 ⁇ l 4 mM S-adenosyl-L-methionine containing 45 nCi S-adenosyl-L- [methyl- 14 C] methionine Amersham
  • 25 ⁇ l enzyme sample e.g. cell free extract
  • the cell extracts typically contain the following activities. Table 1. Methyltran ⁇ fera ⁇ e activitie ⁇ of A. halophila and E. halochloris cell extracts on different substrates.
  • the reaction products were characterized by HPLC.
  • the reaction mixture supernatants a ⁇ de ⁇ cribed above were filtered after centrifugation through a Mini ⁇ art NML 0.2 ⁇ m filter (Sartorius AG) and a 25 - 100 ⁇ l sample was analyzed on AminexHPX-87C cation exchange column (300 x 7.8 mm) (BioRad Laboratories) .
  • the HPLC system used was a Varian 500 equipped with a HP 1047 (B) efractive index detector and a Water ⁇ VISP717 injector. A ⁇ bondapack C 18 -precolumn was u ⁇ ed in the ⁇ y ⁇ tem.
  • Step 1 Ammonium sulphate fractionation. 25 ml of cell free extract (as described in example 1) was diluted to 90 ml and saturated ammonium sulphate in 50 mM Tris-HCl, pH 7.5, was added to achieve 20 % saturation. The solution was incubated for 30 min at 0°C and centrifuged at 15,000 g. The precipitate was discarded and the supernatant purified further.
  • Step 2 Hydrophobic interaction chromatography.
  • the supernatant from step 1 (105 ml) was applied to a Butyl Sepharose 4 FF (Pharmacia) (10 x 50 mm) column pre-equilibrated with 20 % (w/v) ammonium sulphate in 20 mM Tris-HCl, pH 7.5.
  • the column was washed with 45 ml of 20% (w/v) ammonium sulphate in 20 mM Tris-HCl and eluted with a linear gradient of 20-0% ammonium sulphate.
  • the volume of the gradient wa ⁇ 80 ml and the flow rate was 2 ml/ in. Fractions of 3 ml were collected.
  • the active fractions (40 ml) were pooled.
  • the ammonium sulphate was removed by gel filtration (Sephadex G-2S, Pharmacia) .
  • Step 3 Ion exchange chromatography.
  • the sample from step 2 (73 ml) was applied to a DEAE-Memsep 1000 HP (Millipore) (1.4 ml) column pre-equilibrated with 20 mM Tris-HCl, pH 7.5.
  • the column was washed with 15 ml of buffer and eluted with a linear NaCI gradient (0 - 1 M) .
  • the volume of the gradient was 60 ml and the flow rate was 3 ml/min. 2 ml fractions were collected
  • the active fraction ⁇ (8 ml) were pooled and concentrated by ultrafiltration (A icon Centriplus 30; Ultrafree MC 10,000 NMWL filter unit Millipore) to 100 ⁇ l.
  • Step 4 Gel filtration.
  • the concentrated sample from step 3 (100 ⁇ l) was applied to a Superose 12 HR 30 (Pharmacia) column 20 mM Tris-HCl, pH 7.5, containing 150 mM NaCI was u ⁇ ed a ⁇ the elution buffer with flow rate of 0.4 ml/min. 0.5 ml fraction ⁇ were collected.
  • the fraction ⁇ containing glycine-sarcosine methyltransferase (GSMT) activity (1.5 ml) were collected and concentrated by ultrafiltration (Ultrafree MC 10,000 NWML filter unit, Millipore) to 100 ⁇ l .
  • GSMT glycine-sarcosine methyltransferase
  • the purity and the molecular weight were determined by gradient SDS-polyacrylamide gel electrophoresis according to the following procedure. Electrophoresis under denaturing conditions was carried out using pre-made polyacrylamide gel ⁇ lab ⁇ (12 % Tri ⁇ -glycine gel with 4 % stacking gel, Ready Gel ⁇ , Biorad) according to the in ⁇ tructions of the manufacturer. Mid range molecular weight standard from Promega was used. Staining of the gel was performed with 0.25% (w/v) Coomassie Blue R-250 (Promega) in 50 % (v/v) methanol and 10% (v/v) acetic acid. Stained gels were de ⁇ tained with 10% (v/v) methanol and 5% acetic acid (Laemmli, 1970) .
  • the affinity column was prepared as follows. 5 'AMP-Sepharose 4B (Pharmacia) was treated with alkaline phosphata ⁇ e to remove the pho ⁇ phate group of the ligand. The gel wa ⁇ fir ⁇ t ⁇ wollen in water (5 ml of distilled water was u ⁇ ed per 1 g of dry 5 'AMP-Sepharo ⁇ e 4B) . The ⁇ wollen gel wa ⁇ then wa ⁇ hed with 200 ml of di ⁇ tilled water.
  • CIP-buffer 10 mM MgCl 2 , 1 mM dithiothreitol, 50 mM NaCI, 10 mM Tri ⁇ -HCl, pH 7.9
  • the molecular weight of A. halophila SDMT is approximately 32 kDa.
  • the molecular weight was also determined by gel filtration with Superose 12 HR 30 (Pharmacia) column. The flow rate wa ⁇ 0.4 ml/ml.
  • the elution buffer was 20 mM Tris-HCl pH 7.5 containing 150 mM NaCI.
  • the molecular weight was calculated from a calibration curve made with a mixture of standard proteins .
  • the mixture contained 0.5 mg/ml Blue Dextran (0.5 mg/ml), Ferritin (440 kDa), 7.0 mg/ml aldolase (158 kDa), 2.0 mg/ml ovalbumin (43 kDa) and 1.0 mg/ml chymotrypsinogen (25 kDa).
  • the calculated molecular weight wa ⁇ 31.6 kDa, which indicates that the protein is a monomer.
  • the isoelectric focusing wa ⁇ performed with Pharmacia Phast system using gels with pH-gradient from pH 3 to 9 (IEF 3-9) .
  • a mixture of Pharmacia IEF standard proteins with pis from 3.5 to 9.3 were u ⁇ ed a ⁇ standards.
  • the gels were stained by silver staining as described in example 1.
  • the result ⁇ shown in Figure 3 show that the pi of the protein is approximately 4.1-4.2.
  • the activity of the purified protein was determined as described in example 1 with glycine, sarcosine and dimethyl glycine.
  • the data presented in table 2 demonstrates that the isolated protein catalyzes step methylation reaction from sarcosine to dimethyl glycine and from dimethyl glycine to betaine.
  • the pH-optimum of the two methylation reaction ⁇ were determined by u ⁇ ing following buffers: 0.1 M piperazine buffer, pH 5.0; 0.1 M Bis-Tri ⁇ buffer, pH 6.0; 0.1 M Bis-Tris buffer, pH 7.0; 0.1 M Tris-HCl, pH 8.0; 0.1 M Tris-HCl, pH 9.0; 0.1 methanolamine, pH 10.
  • the exact pH value ⁇ of the reaction mixture ⁇ were measured. It can be concluded from Figure 4 that the pH optimum of the both enzyme reactions is at pH 7.5.
  • the temperature dependence of the enzymatic reaction ⁇ were determined with sarcosine and dimethyl glycine. As seen in Figure 5. the temperature optimum is approximately 45 - 50°C. When the temperature is elevated above 50°C, the enzyme is rapidly inactivated.
  • Example 5 In vitro synthesis of betaine by using purified E. halochloris GSMT and A. halophila SDMT
  • the purified GSMT from E. halochlori ⁇ and SDMT from A. halophila were concentrated by ultrafiltration (Ultrafree MC 10,000 NWML filter unit, Millipore) to protein concentrations 4.2 mg/ml and 5.6 mg/ml, respectively.
  • the reaction mixture contained 50 ⁇ l 5.0 mM glycine, 50 ⁇ l 32 mM S-adenosyl methionine in water containing 640 nCi S-adenosyl-L- [methyl- 1 C] -methionine (Amersham) , 50 ⁇ l Buffer II (see example 1), 25 ⁇ l GSMT of E. halochloris and 25 ⁇ l SDMT of A. halophila.
  • the reaction was initiated by adding the enzymes .
  • the reaction mixture wa ⁇ incubated for 2 h at 37°C and the reaction was stopped by adding 150 ⁇ l of charcoal suspension.
  • reaction mixtures were then incubated for 10 min at 0°C and centrifuged for 10 min in a Heraeu ⁇ table top centrifuge.
  • the ⁇ upernatant ⁇ were filtered through Mini ⁇ art NML 0.2 ⁇ m filter (Sartorius AG) .
  • the identification of the reaction products was performed by HPLC as described in example 2.
  • the chromatogram is presented in Figure 6 and it show ⁇ peak ⁇ corresponding to the retention times of sarcosine, dimethylglycine and betaine.
  • N-terminal and tryptic peptide ⁇ peptide ⁇ equences of the purified proteins were determined by using Perkin Elmer/Applied Biosy ⁇ tems Procise 494A protein ⁇ equencing system as described by Kerovuo et al . , 1998.
  • the peptide ⁇ equences obtained are shown in table 3.
  • the genomic DNA from both microbes was isolated essentially as described in Ausubel et al . (1991).
  • the chromo ⁇ omal DNAs were partially digested with Sad and ligated to Sad digested dephosphorylated lambda ZapII arms (Stratagene, La Jolla, California, USA) and packaged to lambda particle ⁇ u ⁇ ing Gigapack III Gold packing extract (Stratagene, La Jolla California, USA) according to protocol provided by manufacturer.
  • the chromo ⁇ omal DNA isolated from the organisms was used as the template DNA in the PCR reactions .
  • the probes were made by PCR using following degenerate primers.
  • the primers were designed according to Sambrook et al. (1989) .
  • the amplification wa ⁇ performed under the following conditions.
  • PCR-fragments obtained were labeled with rediprime DNA labelling sy ⁇ tem (Amersham Life Science) according to the instruction ⁇ given by the manufacturer.
  • the E. halochloris GSMT is encoded by the first ORF of the fragment.
  • the second ORF of E. halochloris clone contains a SDMT gene. This has also been demonstrated by expres ⁇ ing the gene in E. coli (Example 8) .
  • A. halophila SDMT protein is coded by the 3 '-end of the first ORF of the A. halophila clone. The 5 '-end of the same ORF is very homologous to the E. halochloris GSMT.
  • the "betaine operon” codes for a third gene which is homologous to number of S-adenosyl-methionine synthases.
  • the operon ⁇ tructure is schematically shown in Figure 7.
  • the nucleotide and amino acids sequences of the cloned genes have been shown in Figures 8 and 9.
  • the gene coding for the E. halochloris GSMT was amplified by PCR.
  • the purified plasmid used for DNA sequencing in example 6 was used as the template for the PCR reaction.
  • the following primers were used in the PCR reaction:
  • the 3 '-end of the primers are homologous to the 5'- and 3 ' -end of the GSMT gene.
  • the 5 '-end oligonucleotide hybridizes to position 221-241 and the 3 '-end to the position 1001-1024. (See Figure 8) .
  • the primer hybridizing to the 5'- end contains an extra Ncol restriction site such that the nucleotide A at position 224 in Figure 8 i ⁇ replaced by the nucleotide G in the primer and the 3 '-end primer contain ⁇ a Bglll site which were used for cloning.
  • the amplification was performed in the following conditions : 34 cycles of 1 min at 94°C for denaturation. 1 min at 50 °C annealing and 2 min at 72°C for synthe ⁇ i ⁇ .
  • GSMT transformants were grown overnight in 2.5 ml of LB broth containing 100 ⁇ g/ml ampicillin.
  • E. coli XLI E. coli XLI
  • the cell pellet was su ⁇ pended in 100 ⁇ l Buffer II containing 1 mM PMSF (See example 1) and the cell ⁇ were di ⁇ rupted with a MSE Soniprep 150 ⁇ onicator.
  • the cell suspension was ⁇ onicated with ⁇ onication pulses of 5 ⁇ for 10 s .
  • the sample ⁇ were cooled on ice between the pulses .
  • the cell debris was removed by centrifugation at 13,000 rpm for 30 min at 4°C in a Heraeus table top centrifuge.
  • the activitie ⁇ of the supernatants were determined a ⁇ in example 1.
  • the activities using glycine and sarco ⁇ ine a ⁇ substrates were typically 3,000-5,000 dpm/30 min and 1,000-2,000 dpm/30 min., respectively.
  • the strain ⁇ u ⁇ ed in the ⁇ e te ⁇ t ⁇ were the po ⁇ itive clone de ⁇ ignated EGSM and E. coli XLI Blue MRF' tran ⁇ formed with the cloning vector PQE-60.
  • the growth medium u ⁇ ed in this test was the synthetic medium MM63 de ⁇ cribed by Lar ⁇ en et al . (1987) supplemented with 1.5 mil/1 of vitamin solution VA (Imhoff and Tr ⁇ per, 1977) and 100 ⁇ l/ml ampicillin.
  • the bacterial strain ⁇ were grown to mid-exponential growth phases with shaking at 180 rpm at 37°C and centrifuged (1,000 g, 15 min) .
  • the cells were resu ⁇ pended in the growth medium to ab ⁇ orbance of 0.9 at 600 nm.
  • the gene encoding the E. halochloris SDMT was amplified by PCR.
  • the purified plasmid used for DNA sequencing in example 6 was used a ⁇ the template for the PCR reaction.
  • the following primer ⁇ were used in the PCR reaction:
  • the primers are homologous to the 5'- and 3 ' -end of the E. halochloris SDMT gene.
  • the 5' -end oligonucleotide hybridizes to position 1031-1054 and the 3 '-end to the position 1844- 1867 ( Figure 8) .
  • the primer hybridizing to the 5' -end contains an extra Ncol restriction site and the 3 '-end primer a Bglll site which were used for cloning of the fragment.
  • the cultivations and preparation of the cell-free extracts were performed essentially as described in example 7.
  • the ⁇ onication of the cell extract pulses was shortened to 3 x 2 second interval ⁇ (total sonication time was 6 s).
  • the activities using sarcosine and dimethyl glycine as substrate ⁇ were typically 20,000 dpm/30 min. with both ⁇ ub ⁇ trate ⁇ .
  • the DNA construct made for this experiment contain ⁇ both GSMT and SDMT genes separated by a short (3 nucleotides long) linker.
  • the DNA fragment was obtained by amplification of the purified pla ⁇ mid used for DNA sequencing in example 6.
  • the primers are homologous to the 5 '-end of the E. halochloris GSMT and the 3 ' -end of the E. halochloris SDMT gene.
  • the 5 '-end oligonucleotide hybridizes to position 221- 241 and the 3 ' -end to the position 1844-1867 (See Figure 8) .
  • the primer hybridizing to the 5 '-end contains an extra Ncol restriction site and the 3' -end primer a Bglll site.
  • the enzymatic activities were as ⁇ ayed as described in example I .
  • the cell extracts of the transformant ⁇ clearly ⁇ howed activity with glycine, ⁇ arcosine and dimethylglycine.
  • the activities of the cell extracts with the three substrate ⁇ were all over 20,000 dpm/30 min.
  • the test was performed es ⁇ entially a ⁇ described in example 7.
  • the po ⁇ itive clone de ⁇ ignated EhFU was used in this te ⁇ t.
  • the growth medium used in this test was the synthetic medium MM63 described by Larsen et al. (1987) ⁇ upplemented with 1.5 ml/1 of vitamin solution VA (Imboff and Tr ⁇ per, 1977) and 100 ⁇ l/ml ampicillin.
  • the medium contained 1% (wlv) glucose.
  • the clone EhFU ( Figure 10) and the control strain (E coli XLl-Blue MRF' transformed with the cloning vector PQE-60) were grown to mid-exponential pha ⁇ e with ⁇ haking at 180 rpm at 37°C.
  • the cell ⁇ were centrifuged at 1,000 g for 10 min and resuspended in the growth medium ⁇ o that the turbidity goot was 0.640. 5 ml of this cell suspension wa ⁇ inoculated to 50 ml of media containing 0.22 or 0.33 M NaCI and 25 mM L-methionine.
  • the bacterial ⁇ train ⁇ were grown for 2 h with ⁇ haking at 180 rpm at 37°C and 1 mM IPTG was added.
  • the cell pellets were suspended in 2 ml of water and kept in a boiling water bath for 10 min.
  • the su ⁇ pen ⁇ ion was centrifuged for 15 min at 23,000 g and the supernatant collected and the pellet resuspended in water. This extraction was repeated twice.
  • the three supernatants were combined.
  • the volumes of supernatant ⁇ were measured and the supernatant ⁇ were filtered and analyzed by HPLC as described in example 1.
  • the betaine produced inside the cells is presented in table 4.
  • Example 10 Expression of the DNA fragment encoding the protein isolated as A. halophila SDMT in E. coli
  • the gene sequencing results revealed that a single gene codes for the A halophila GSMT and SDMT.
  • the fusion protein was not, however, successive ⁇ fully purified from the A. halophila cell extract ⁇ . Instead a protein with SDMT activity was isolated.
  • the corresponding part of the GSMT-SDMT gene i ⁇ expres ⁇ ed in E. coli.
  • the gene fragment encoding the SDMT enzyme activity was amplified by PCR.
  • the genomic DNA from A. halophila isolated in example 6 was used as the template for the PCR reaction.
  • the following primers were used in the amplification:
  • the primers are homologous to the 5'- and 3 '-end of the ASDMT gene.
  • the 5' -end oligonucleotide hybridizes to position 1048- 1068 and the 3' -end to the position 1879-1902 (See Figure 9) .
  • the primer hybridizing to the 5 '-end contains an extra Ncol restriction site and the 3 '-end primer a Bglll site.
  • Example 11 Expression of A. halophila GSMT-SDMT fusion protein in E. coli
  • the primer ⁇ are homologous to the 5'- and 3 ' -end of the AGSMT-ASDMT gene.
  • the 5 '-end oligonucleotide hybridizes to position 208-231 and the 3 '-end to the position 1879-1902 (See Figure 9).
  • the primer hybridizing to the 5 '-end contains an extra Ncol restriction site ⁇ uch that the nucleotide A at po ⁇ ition 211 in Figure 9 i ⁇ replaced by the nucleotide G in the primer and the 3 '-end primer contain ⁇ a Bglll ⁇ ite.
  • the purified pla ⁇ mid used for DNA-sequencing in example 6 was used as a template for the PCR reaction.
  • the amplification was performed in following conditions: 34 cycles of 30 s at 94°C for denaturation, 1 min at 50°C annealing and 2 min at 72°C for ⁇ ynthesis.
  • Ligation of the amplification product into Ncol/Bglll cut PQE-60 and the transformation of XL-1 Blue MRF' cells was performed as in example 7.
  • the induction and preparation of the cell-free extracts was performed essentially as in example 9 except that the sonication pulses were shortened to 2 ⁇ and the total ⁇ onication time to 6 s .
  • the cell-free extract was analyzed by SDS-polyacrylamide gel electrophoresis as in example 2.
  • the pellet from the centrifuged suspension wa ⁇ suspended to 10 mM Tri ⁇ -HCl-buffer, pH 8.0 containing 8 M urea and 0.1 M Na3P0 4 to ⁇ olubilize the proteins of the pellet and centrifuged for 15 min in a Heraeus table top centrifuge at 13,000 rpm.
  • the ⁇ upernatants were analyzed by SDS-polyacrylamide gel electrophoresis as in example 2.
  • the enzymatic activities were assayed as described in example 1.
  • the cell extracts of the transformant ⁇ clearly showed SDMT activity.
  • the activities on sarcosine and dimethyl glycine were typically 10,000 dpm/30 min and 20,000 dpm/30 min., respectively. There was no activity on glycine.
  • the SDS-polyacrylamide gel of the cell-free extract showed no major protein band of correct ⁇ ize.
  • the insoluble pellet solubilized with 8 M urea showed a major band corresponding to the molecular weight of the GSMT-SDMT fusion protein.
  • the results indicate that when A. halophila GSMT- SDMT is over-expres ⁇ ed in E. coli it forms inclusion bodies.
  • a fraction of the protein - which corresponds the SDMT - is proteolytically cleaved and remains soluble in the cells .
  • Example 12 Expression of E. halochloris GSMT and SDMT in tobacco and potato
  • Tobacco and potato plants can be transformed by AgrroJacteriu-n mediated tran ⁇ formation system.
  • Identical DNA construct can be used for both plants.
  • the GSMT gene is first tran ⁇ formed into the plant u ⁇ ing a pla ⁇ mid containing a kanamycin re ⁇ istance marker. Positive transgenic plants obtained by screening for the enzyme activity are then used as host plants for second transformation of the SDMT gene. Another ⁇ election marker, hygromycin selection is used in the second transformation. Experiments are performed using stable transformants of the F x generation.
  • the genes of E. halochloris GSMT and SDMT are amplified by PCR by using plasmid pEFU (see example 10) as the template.
  • the primers used hybridize to the same regions of the DNA as shown in Fig. 8 (GSMT: primer 1 and primer 2; SDMT; primer 3 and primer 4) .
  • the final DNA constructs are made using suitable restriction sites to transfer the genes to plant transformation vectors.
  • PBin 19 based pGPTV vectors (Becker et al, 1992) are used which have a strong 35S promoter and the CaMv polyadenylation signal.
  • Strain EHA 105 Hood, E.E. et al . , (1993) is used as a vector to transform tobacco basically as described by Rogers et al . (1986).
  • Strain ClC58p-GV3850 Zambryski et al . , (1983); Van Larabece et al . , (1974) is used a ⁇ an alternative host to transform potato Solanum tuherosum (Desire) according to Dietze et al . (1995) .
  • the transformants are analyzed by Southern blot analysis to check for the presence of the genes. PCR-amplified, DIG- labelled (Boehringer) 200 bp gene fragments are used as a probe. The enzymatic activities of the cell extracts of transgenic plant ⁇ and the levels of ⁇ arcosine, dimethyl glycine and betaine are analyzed as described in Example 1.
  • Stress tolerance for example, tolerance to drought, salinity, cold or freezing, resistance to pathogens, etc., is determined according to methods known in the art, for example, methods described in the technological background section of the present application.
  • Example 13 Expression of E. halochloris GSMT and SDMT in rice
  • the plasmid construction ⁇ described in Example 13 are also used to transfer the GSMT and SDMT genes to rice by particle bombardment.
  • the GSMT are transferred to rice first and positive regenerated tran ⁇ formants are used as host plants for the SDMT transformation.
  • Immature Oryza sativa embryos of the Japonica variety Taipei 309 are aseptically isolated 10-14 days after pollination from greenhouse plants and plated scutulum site up on solid MS medium (Murashige and Skoog, 1962) containing 3% sucrose, 2 mg/1 2,4- dichlorophenoxyacetic acid and 50 mg/l cefotaxime (MSI) . After 4-6 days (28°C, darkness) embryos are transferred to ⁇ olid MS medium containing 10% ⁇ ucro ⁇ e, 2 mg/1 2,4- dichlorophenoxyacetic acid and 50 mg/1 cefotaxime (MS2) and ⁇ ubjected within 1 hour to microprojectile bombardment with a particle inflow gun.
  • solid MS medium Morashige and Skoog, 1962
  • MSI cefotaxime
  • the DNA fragment containing the mehtyltransferase gene and the selective marker (5 ⁇ g) is precipitated on 1-3 mm gold particles (Aldrich) as described by Vain et al . , (1993) .
  • Gold particles 400 mg per bombardment
  • Embryo ⁇ are placed 16 cm below the ⁇ yringe filter. Twenty four hour ⁇ po ⁇ t-bombardment embryos are subjected to selection on solid media (containing hygromycin or kanamycin) and incubated at 28°C in the dark.
  • Developing calli are isolated 3 to 6 weeks later, and transferred to a callus increa ⁇ ing media (R2 medium supplemented with: 6% ⁇ ucrose, MS vitamins, 100 mg/l inositol, 2 mg/l 2, 4-dichlorophenoxyacetic acid, 50 mg/l cefotaxime and 20 mg/l hygromycin B kanamycin) .
  • the calli are incubated in thi ⁇ media at 28°C in the dark and ⁇ ubcultured weekly.
  • Re ⁇ i ⁇ tant calli are tran ⁇ ferred to solid R2 regeneration media supplemented with 2% sucrose, 3% sorbitol, 20 mg/l hygromycin B, 1 mg/l zeatin, 0.5 mg/l indole-3-acetic acid, MS vitamins and 0.65% agarose.
  • the callus tissue is maintained at 28°C with 12 h of light in order to enhance shoot formation.
  • the calli are then subcultured every 3 week ⁇ until ⁇ hoot ⁇ had reached a length of 2-3 cm. They are transferred to half-strength MS rooting medium without hormones, supplemented with 1.5% sucro ⁇ e and 0.3% gelriteR (Sigma) .
  • plantlets are tran ⁇ ferred directly to the green-house and planted in soil. Plantlets are grown in 7 liter aquaculture pot ⁇ with fertilizer enriched earth, 3 plants per pot (day: 12 h, 28°C, 80% humidity; night: 12h, 21°X, 60% humidity) until they flower and set seeds.
  • Southern blot analy ⁇ i ⁇ is performed as described previously (Burkhardt et al., 1997).
  • a PCR amplified, DIG-labelled (Boehringer) 200-bp fragment of the coding region of the GSMT or SDMT genes is used as a probe .
  • Stress tolerance for example, tolerance to drought, salinity, cold or freezing, resistance to pathogens, etc., is determined according to methods known in the art, for example, methods described in the technological background section of the present application.
  • Example 14 Expression of the E. halochloris GSMT and SDMT in yeast
  • pYX242 plasmid (R&D systems, USA) was used for expressing the GSMT and SDMT genes in Saccharomyces cerevisiae .
  • the plasmid used (pYX242) is a E. coli -Saccharomyces cerevisiae ⁇ huttle vector containing a bacterial origin of replication and ampicillin re ⁇ i ⁇ tance gene, a yeast ⁇ S. cerevisiae) origin of replication from 2 ⁇ m DNA, and the yeast LEU2 gene for selection in yeast.
  • the two genes are expressed under the yeast triose phosphate isomerase (TPI) promoter.
  • TPI yeast triose phosphate isomerase
  • the DNA of the pla ⁇ mid pEFU described in Example 10 wa ⁇ used as the template of PCR reactions.
  • the primers u ⁇ ed hybridize to DNA ⁇ equence ⁇ shown in Fig. 8 (primer 1 and primer 4) and thus amplify both the GSMT and SDMT genes.
  • the PCR fragment was ligated to the promoter of the expression plasmid with standard methods. A fragment containing a TPI transcription terminator and a fragment containing the TPI promoter was ligated between the two genes. Thus, both gene ⁇ are expressed under the TPI promoter.
  • the primers used in the amplification of the fragments ligated in the expression plasmid contained suitable restriction sites that were used in the cloning. S.
  • the transformants were grown in YNB-medium supplemented with amino acid mixture without leucine (R &D systems product manual) .
  • the cultivation was done overnight at 30°C by ⁇ haking at 180 rpm.
  • 5 ml of culture supernatant was centrifuged (1,700 g, 10 min) and the cell pellet wa ⁇ suspended in 200 ⁇ l of the assay buffer (see example 1) supplemented with 1 mM PMSF.
  • the cells were broken by vortexing with glass beads (10 x l min intervals) .
  • the cells were kept on ice between the pulses.
  • the cell debris was centrifuged down (30 min, 4°C, 10,000g).
  • the methylase activities were assayed from the supernatant as described in example 1.
  • the relative enzyme activities on different substrates were the following: glycine - 17,000 dpm/30 min; sarco ⁇ ine - 71,000 dpm/30 min and dimethyl glycine - 530,000 dpm/30 min.
  • JP patent 87,190,078 Kojima, Y., Aisui, S., Ando, M. (1987) Kapper, R., Kempf, B., Bremer, E., (1996) J. Bacteriol. 178, 5071-5079

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Abstract

The present invention relates to proteins which are capable of functioning as methyltransferases. More, specifically, the present invention relates to methyltransferases which are capable of carrying out at least one of the following reactions: the conversion of glycine to sarcosine, sarcosine to dimethyl glycine and dimethyl glycine to betaine in the presence of a methyl group donor. Furthermore, the present invention relates to nucleic acid molecules encoding such methyltransferase proteins, recombinant organisms which are capable of expressing said nucleic acids as well as the use of said recombinant organisms.

Description

Methyltransferases, nucleic acid molecules encoding methyltransferases, their recombinant expression and uses thereof
The present invention relates to proteins which are capable of functioning as methyltransferases. More, specifically, the present invention relates to methyltransferases which are capable of carrying out at least one of the following reactions : the conversion of glycine to sarcosine (N- methylglycine) , sarcosine to N,N-dimethyl glycine and N,N- dimethyl glycine to betaine (N,N,N-trimethylglycine) in the presence of a methyl group donor. Furthermore, the present invention relates to nucleic acid molecules encoding such methyltransferase proteins, recombinant organisms which are capable of expressing said nucleic acids as well as the use of said recombinant organisms .
Technological background
Betaine (N,N,N-trimethylglycine) is a quaternary ammonium compound which can be found in many micro-organisms, animals and plants . Betaine is synthesized or accumulated in living cells in response to abiotic stress (salinity, desiccation or low temperatures) (Mc Gue and Hanson, 1990; Csonka, 1989; Yancey et al . , 1982; Wyn Jones et al . , 1977; Gorham, 1995; Bohnert and Jensen, 1996) . Due to its physical properties, betaine is an osmolyte and thus it is able to restore and maintain osmotic balance of living cells. In addition, it has been demonstrated that betaine stabilizes and protects cell membranes (Coughlan and Heber, 1982) and other macromolecules (i.e. enzymes) in the cell (Papagiorgiou and Murata, 1995) .
Betaine is synthesized by a number of microbes. In addition, these microbes are usually capable of accumulating betaine or a precursor, choline, from the culture medium (Boch et al., 1994; Perroud and Rudulier, 1985; Kempf and Bremer, 1995; Glaasker et al., 1996; Peter et al . , 1996; and Kappes et al., 1996) . Practically all halophilic bacteria are able to use betaine as at least one of their osmolytes in order to survive in the high ionic strength environment .
Also, many plants synthesize betaine in response to drought or salinity (Rhodes and Hanson, 1993; Gorham, 1995). A correlation between cold tolerance and betaine synthesis has been demonstrated (Holmberg, N. 1996, Kishitani et al . , 1994; Nomura et al., 1995). Intracellular concentrations as low as 1 mM have shown to give protective effects in plants (Ishitani et al . , 1993). Increasing the betaine content of plants by genetic engineering or plant breeding has shown to result in more salt or cold tolerant transgenic plants. A betaine biosynthesis pathway has been introduced, e.g. in tobacco (Lilius et al . 1996; JP 8-266179; JP 8-103267) Arabidopsis (Hayaεhi et al . , 1997) and rice (Nakamura et al . , 1997; Guo, 1997) . However, the results obtained are very preliminary and only phenotypic effects (salt or cold tolerance) have been demonstrated. Very little is known with respect to the relationship between the concentration dependence of the intracellular betaine and stress tolerance.
It has recently been shown that plants can acquire better stress tolerance by accumulating exogenously applied betaine. Foliar spraying of betaine in a specific phase of growth has been shown to increase the productivity of many crops . (Makela et al., 1996; Agboma, 1997). Typically, 15 % crop yield improvements have been obtained with many species (sorghum, maize, soybean, cotton, potato, tomato) under conditions of salinity and drought. The detailed physiological mechanism of betaine action is not fully understood, but it is known that betaine stimulates photosynthesis and decreases photorespiration.
In animal cells, betaine also acts as a methyl group donor. The most important function is to the ability to methylate homocysteine back to methionine, which can then further be used as a methyl group donor when metabolized to S-adenosyl methionine (SAM) . It has been demonstrated that orally administered betaine relieves diarrhea and dehydration in many animals and inhibits invasion of gut epithelium by coccidia parasite (Ferket, 1994; Augustine and McNaughton, 1996) . As a methyl group donor betaine has been shown to be lipotropic, thus decreasing the amount of fat in chicken meat (Saunderson and MacKinlay, 1990; Barak et al . , 1993) .
The most extensively studied betaine biosynthesis pathway is the two-step oxidation reaction of choline to betaine via betaine aldehyde. This metabolic pathway has been demonstrated to exist in number of microbes, plants and animal cells. The choline-betaine pathway of E. coli (La ark et al., 1991) and Pseudomonas aeruginosa (Nagasawa et al . , 1976) comprises an oxygen-dependent choline dehydrogenase, which catalyzes the oxidation of both choline to betaine aldehyde and betaine aldehyde to betaine. The E. coli choline dehydrogenase gene has been cloned and sequenced. In addition, it has successfully been expressed in many heterologous organisms (Nomura et al . , 1995; Lilius et al., 1996; Hayashi et al . , 1997; Nakamura et al . , 1997; Guo, 1997) . The enzyme is membrane-bound. The reaction is independent of soluble cofactors and electron-transfer linked. Choline dehydrogenase genes from Sinorhizobium meliloti (Pocard et al . , 1997) and Bacillus (Boch et al., 1996) have also been isolated.
Alternatively, the oxidation of choline to betaine can be catalyzed by a choline oxidase found for example in some Corynebacteria, Brevibacterium and Alcaligenes species
(Nakanishi and Machida, 1981; Kojima et al . , 1987). The enzyme has been shown to also exist in some fungal strains
(Tani et al . , 1979). The choline oxidase of Arthrobacter pascens has been cloned and successfully expressed in E. coli
(Rozwadovski et al . , 1991) or Synechococcus (Deshnium et al . , 1995) . The reaction uses molecular oxygen as the hydrogen acceptor and hydrogen peroxide is formed in the reaction. In plants, the synthesis of betaine pathway has been investigated in detail in sugar beet and spinach (McCue et al., 1992; Weretilnyk and Hanson, 1989). The first step is catalyzed by a choline mono-oxygenase. In plants the enzyme is located in the chloroplast stroma (Brouquisse et al., 1989) . The gene has recently been cloned from spinach (Rathinasabapathi et al., 1997).
The second oxidation step from betaine aldehyde to betaine may also be catalyzed by a betaine aldehyde dehydrogenase. A betaine aldehyde dehydrogenase has also been found in a number of organisms (Pseudomonas aeruginosa (30) ) . Also some plants have this enzyme (Hanson et al., 1985) . In plants, the enzyme has been demonstrated to have wider substrate specificity and thus it also catalyzes other reactions (Trossat et al . , 1997) . The gene has been cloned from E. coli (Lamark et al., 1991), spinach (Weretilnyk and Hanson, 1990) and also from barley (Ishitani et al . , 1995) . The E. coli gene has also successfully been expressed in transgenic tobacco (Holmstrόm et al . , 1994).
Ability to synthesize betaine de novo is rare among aerobic heterotrophic eubacteria. Of all strains examined, only Actinopolyspora halophila and a related isolate have been shown to produce betaine from simple carbon sources (Severin et al., 1992; Galinski, 1993). There are few examples of other organisms which have been shown to be able to synthesize betaine from simple carbon sources. The data is usually based on metabolic studies using NMR. Roberts and co-workers (1992) have shown that some archaebacterial methanogens (Methanohalophilus) could synthesize betaine from glycine via a methylation reaction. However, no enzymes catalyzing the reactions have successfully been isolated from these organisms. A similar pathway has been suggested to exist in Ectothiorhodospira halochloris (Galinski and Truper, 1994) . Attempts to characterize the glycine biosynthesis pathway in Ectothiorhodospira halochloris have been made (Tschichholtz-Mikus, 1994) . According to the hypothesis proposed by this author, betaine would be synthesized from glycine by three methyltransferases, each specific for one methylation reaction. The isolation of the purified enzymes was, however, not successful and only one enzyme, specified as dimethyl glycine methyltransferase was partially purified. In addition, the methodology used to study reactions was rather simple and as demonstrated herein, the results obtained by the group differ from those obtained by the present inventors .
Betaine is used as feed additive in feed industry. Thus, transgenic plants producing high amounts of betaine in vivo would have better nutritional value. Feed crops (e.g. maize or soybean) producing sufficient amounts of betaine could therefore directly be used in feed without the need of betaine supplementation.
Although betaine is synthesized by many plants, there are several commercially important crops such as potato, rice, tomato and tobacco which do not accumulate betaine. For example, Bulow and co-workers (1995) were the first to demonstrate that expression of the E. coli choline dehydrogenase in tobacco improves the salt tolerance and freezing tolerance (Holmberg, 1996) of transgenic potato and tobacco due to endogenously synthesized betaine. The same phenomenon has been demonstrated also with Arabidopsis (Hayashi et al., 1997) and rice (Nakamura et at., 1997; Guo, 1997) . Therefore, expression of the methyltransferases in plants can facilitate stress tolerance and improve the productivity of the plants when grown under conditions of water stress or freezing and cold temperatures.
Moreover, betaine has shown to induce pathogenesis-related protein expression in plants (Xin et al . , 1996) as well as increasing the resistance of plants to attack by pathogenic fungi or nematodes (Blunden et al . , 1996; Wu et al . ,1997) and may decrease the incidence of nematode (e.g. Meloidogne javanica and M. incognita) attack in plants. Therefore, transgenic plants producing endogenous betaine, can be more resistant to fungal pathogens. Moreover, the betaine synthesis in the plants may be coupled to the systemic resistance genes which are induced when the plant is attacked by pathogens .
Endogenously synthesized betaine may also affect the viability of microbes and therefore it would improve their performance in various biotechnical processes. For instance, in high cell density fermentation or immobilized cell systems, the production microbes are subjected to considerable environmental stress. Betaine has successfully been used in fermentation media to increase the product yield in amino acid production. For instance, betaine has shown to relieve stress and improve yield of lysine producing Brevibacterium lactofermentum (Kawahara et al . , 1990). Betaine is also commercially sold for the purpose (Nutristim®, Cultor Corp) . Thus, endogenously synthesized betaine can improve productivity in biotechnical processes where the cells are subjected to abiotic stress.
Microbes also suffer from stress when subjected to high temperatures or when cells are freeze-dried or frozen. The viability of yeast or bacterial cells may be dramatically reduced in these processes used in e.g. frozen dough manufacturing or preservation of lactic acid bacterium starters. Therefore, it would be highly advantageous if one could improve the viability of microbes subjected to freeze-thaw or freeze-drying processes by accumulating betaine inside the cells. In addition, it has been shown that exogenously applied betaine improves the viability of microbes in extreme pH (Smirnova and Oktyabrsky, 1995; Chambers and Kunin, 1985) . Improved performance of beneficial, probiotic microbes organisms in animal digestive tract can be utilized in animal nutrition. Thus, introduction of a betaine synthesis pathway can improve the stress tolerance of "probiotic" lactic acid microbes which efficiently bind to gut epithelium in cells, providing a way to balance the microbial population in the GI tract and to improve pathogen resistance.
Betaine has been shown to stabilize proteins in the cells . For example, it has also been demonstrated that cytoplasmic accumulation of betaine will reduce the formation of inclusion bodies (Blackwell and Horgan, 1991; Bhandari and Gowrishankar, 1997) which is a problem often encountered when heterologous proteins are expressed in E. coli. Thus, co-expression of the genes of betaine biosynthesis with the protein of interest should result in better solubility of the heterologous protein and reduce the amount of inclusion bodies .
The use of the glycine methylation pathway may have a number of advantages over the oxidative synthesis from choline. Glycine is synthesized by practically all organisms and as an amino acid, this metabolite is present in high concentrations in the cells. In contrast, the availability of intracellular choline may limit betaine biosynthesis. In addition, the metabolism and formation of glycine in cells is known and the genes of this metabolic pathway have been cloned, thus allowing the engineering of the glycine pathway.
Based on the above, an object of the present invention is to provide proteins which are capable of acting in a biosynthetic pathway from glycine to betaine as well as methods for the purification and production of said proteins.
A further object of the present invention is to provide nucleic acid molecules which, when transformed into a host organism, encode proteins which are capable of acting in a biosynthetic pathway from glycine to betaine. A further object is to provide recombinant microorganisms which are capable of expressing one or more proteins which are capable of acting in a biosynthetic pathway from glycine to betaine for the production of betaine and precursors thereof .
Furthermore, an object of the present invention is to provide recombinant plants which express one or more proteins which are capable of acting in a biosynthetic pathway from glycine to betaine for the production of betaine and precursors thereof .
Furthermore, an object of the present invention is to provide a method for the production of betaine and precursors thereof, for example sarcosine and dimethyl glycine, in recombinant organisms .
A further object is to provide recombinant organisms which have an increased concentration of intracellular betaine and are useful in the fields of recombinant heterologous protein production, agriculture, etc.
A further object of the present invention is to provide nucleic acid probes and method for identifying and cloning genes which encode proteins which are capable of participating in a biosynthetic pathway from glycine to betaine.
A further object of the present invention is to provide methods for improving the general growth and/or productivity of an organism including enhancing stress tolerance, for example, salt tolerance, freezing tolerance and cold tolerance, enhancing resistance to drought, water stress and attack by pathogens in organisms.
Furthermore, an object of the present invention is to provide recombinant microorganisms which have improved viability in culture, enhanced pH tolerance in culture, result in decreased inclusion body formation when expressing a heterologous protein, result in increased solubility, stability and/or yield of a heterologous protein expressed in said organism.
Moreover, it is an object of the present invention to provide an animal feed and animal feed ingredient having enhanced nutritional value.
Other objects of the present invention will be apparent to the skilled person based on the information provided herein.
Summary of the Invention
The inventors have identified, isolated and purified proteins which are capable of carrying out at least one of the following reactions in a metabolic pathway from glycine to betaine: the conversion of glycine to sarcosine (N- methylglycine) , sarcosine to N,N-dimethyl glycine and N,N- dimethyl glycine to betaine (N,N,N-trimethylglycine) in the presence of a methyl group donor. These proteins are designated herein as methyltransferases based on their ability to transfer a methyl group from a methyl group donor to a methyl group acceptor.
The above mentioned reactions are individual steps in a three-step methylation reaction pathway of glycine to betaine in certain microorganisms, for example, Ectothiorhodospira halochloris and Actinopolyspora halophila.
For example, a methyltransferase of the present invention (designated hereinafter as glycine-sarcosine methyltransferase or GSMT) has been isolated from Ectothiorhodospira halochloris and Actinopolyspora halophila which is capable of catalyzing methylation reactions that convert glycine to dimethyl glycine, i.e. convert glycine to sarcosine (N-methyl glycine) and sarcosine to dimethyl glycine (N,N-dimethyl glycine) .
As a further example, a methyltransferase according to the present invention (designated hereinafter as sarcosine- dimethylglycine methyltransferase or SDMT) has been isolated from Ectothiorhodospira halochloris and Actinopolyspora halophila which is capable of catalyzing methylation reactions that convert sarcosine to betaine, i.e. convert sarcosine (N-methyl glycine) to dimethyl glycine (N,N- dimethyl glycine) and dimethyl glycine to betaine.
The activities of GSMT and SDMT are described below.
glycine
g m
N-methyl glycine (sarcosine)
glycine-sarcosine me- methyltransferase lycine (GSMT) sferase
N,N-dimethyl glycine
ne- ycine ferase
N,N,N-trimethyl glycine (betaine) The methyltransferases of the present invention are capable of utilizing S-adenosyl methionine (hereinafter also referred to as SAM) as a methyl group donor in the above reactions.
Brief Description of the Figures
Figure 1. Formation of methylation products from glycine, sarcosine and dimethyl glycine substrates using the A . halophila cell extract. The retention times of the standards are shown by arrows .
Figure 2. Analysis of purified methyl transferases on SDS- PAGE. A) E. halochloris GSMT; B) A. halophila SDMT. Lane 1, Purified protein sample; lane 2, molecular weight marker.
Figure 3. The determination of the isoelectric point of A . halophila SDMT by isoelectric focusing.
Figure 4. The pH-optimum of A . halophila SDMT. (•) Activity on sarcosine; (A) Activity on dimethyl glycine
Figure 5. The temperature dependence of A. halophila SDMT activity. (•) Activity on sarcosine; (A) Activity on dimethyl glycine.
Figure 6. In vi tro synthesis of betaine by using the purified E. halochloris GSMT and A. halophila SDMT enzymes. The retention times of the standards are shown by arrows.
Figure 7. The schematic structure of the betaine operons of A . halophila and E. halochloris . GSMT; glycine sarcosine methyltransferase. SDMT; sarcosine dimethyl glycine methyltransferase. SAMS; S-adenosyl methionine synthase.
Figure 8. The nucleotide and amino acid sequence of the E. halochloris betaine operon. The arrows indicate the amino acids encoding GSMT, SDMT and SAMS. The underlined regions indicate regions which are hybridized with the primers used to construct the expression vectors in heterologous organisms. The * indicates a stop codon.
Figure 9. The nucleotide and amino acid sequence of the A . halophila betaine operon. The underlined regions indicate regions which are hybridized with the primers used to construct the expression vectors in heterologous organisms. The * indicates a stop codon.
Figure 10. Schematic presentation of the expression plasmid used in expression of the methyl transferases. The insert was ligated to vector digested with Ncol/Bgrlll .
Figure 11. The growth curves of E. coli transformants carrying the E. halochloris GSMT gene (EGSM) . Transformant carrying only the cloning vector (PQE-60) was used as the control .
Figure 12. The growth curves of E. coli transformants carrying the E. halochloris GSMT and SDMT genes (EhFU) . Transformant carrying only the cloning vector (PQE-60) was used as the control.
Detailed Description of the Invention
One embodiment of the present invention provides a methyltransferase, for example GSMT, capable of catalyzing the conversion of glycine to sarcosine (Ν-methyl glycine) and/or the conversion of sarcosine to dimethyl glycine (Ν,Ν- dimethyl glycine) . Preferably, said methyltransferase comprises an amino acid sequence selected from the group consisting of:
(a) an amino acid sequence as depicted in SEQ ID NO: 2 and an amino acid sequence as depicted in SEQ ID NO: 6,
(b) a fragment of an amino acid sequence as defined in (a) and (c) a derivative of an amino acid sequence as defined in (a) and (b) .
A fragment of an amino acid sequence as depicted in SEQ ID NO: 2 or SEQ ID NO : 6 is designated as any fragment of an amino acid sequence as depicted in SEQ ID NO: 2 or SEQ ID NO: 6 which is capable of catalyzing the conversion of glycine to sarcosine (N-methyl glycine) and/or the conversion of sarcosine to dimethyl glycine (N,N-dimethyl glycine) .
A derivative of an amino acid sequence as depicted in SEQ ID NO: 2 or SEQ ID NO: 6 is designated as any mutation, deletion, addition, substitution, insertion or inversion of one or more amino acids in the of amino acid sequence as depicted in SEQ ID NO: 2 or SEQ ID NO : 6 which is capable of catalyzing the conversion of glycine to sarcosine (N-methyl glycine) and/or the conversion of sarcosine to dimethyl glycine (N,N-dimethyl glycine) . Preferably, a derivative of a methyltransferase which is capable of catalyzing the conversion of glycine to sarcosine (N-methyl glycine) and/or the conversion of sarcosine to dimethyl glycine (N,N-dimethyl glycine) has about 60 % homology, preferably about 70 % homology, more preferably about 80 % homology, and most preferably about 90 % homology to the corresponding amino acid sequence depicted in SEQ ID NO : 2 or SEQ ID NO: 6. Preferably, when the above amino acid sequences has one or more mutations, substitutions, additions and/or insertions, the amino acids constituting these changes are selected from the 20 standard naturally-occurring amino acids found in proteins, i.e. Ala, Val, Leu, lie, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, Asp, Glu, Lys , Arg and His. Preferably, the mutation (s) and/or substitution (s) are conservative, for example, Ala, Val, Leu, lie, Pro, Phe, Trp, or Met residue (s) are replaced with one of these amino acids, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, residue (s) are replaced with one of these amino acids , Asp or Glu are replaced with one of these amino acids and Lys, Arg or His are replaced with one of these amino acids . In a preferred embodiment, a methyltransferase according to the invention has the amino acid sequence depicted in SEQ ID NO: 2.
In another preferred embodiment, a methyltransferase according to the invention has the amino acid sequence depicted in SEQ ID NO: 6.
The methyltransferase of the present invention capable of catalyzing the conversion of glycine to sarcosine (N-methyl glycine) and/or the conversion of sarcosine to dimethyl glycine (N,N-dimethyl glycine) can exist in the form of an active enzyme or as a zymogen. The term 'zymogen' designates a protein molecule or fragment or derivative thereof (as defined above) which is synthesized in an inactive form and is capable of being activated in vitro or in vivo by the chemical or enzymatic cleavage of one or more peptide bonds . A preferred zymogen of the methyltransferase of the present invention capable of catalyzing the conversion of glycine to sarcosine (N-methyl glycine) and/or the conversion of sarcosine to dimethyl glycine (N,N-dimethyl glycine) comprises the amino acid sequence as depicted in SEQ ID NO: 2 and SEQ ID NO: 3, wherein the N-terminus of SEQ ID NO: 3 is joined to the C- erminus of SEQ ID NO: 2.
A further embodiment of the present invention provides a methyltransferase, for example SDMT, capable of catalyzing the conversion of sarcosine (N-methyl glycine) to dimethyl glycine (N,N-dimethyl glycine) and/or dimethyl glycine to betaine. Preferably, said methyltransferase comprises an amino acid sequence selected from the group consisting of :
(a) an amino acid sequence as depicted in SEQ ID NO: 3 and an amino acid sequence as depicted in SEQ ID NO: 7,
(b) a fragment of an amino acid sequence as defined in (a) and
(c) a derivative of an amino acid sequence as defined in (a) and (b) . A fragment of an amino acid sequence as depicted in SEQ ID NO: 3 or SEQ ID NO: 7 is designated as any fragment of an amino acid sequence as depicted in SEQ ID NO: 3 or SEQ ID NO: 7 which is capable of catalyzing the conversion of sarcosine (N- methyl glycine) to dimethyl glycine and/or the conversion of dimethyl glycine to betaine.
A derivative of an amino acid sequence as depicted in SEQ ID NO: 3 or SEQ ID NO: 7 is designated as any mutation, deletion,- addition, substitution, insertion or inversion of one or more amino acids, or combination thereof, in the of amino acid sequence as depicted in SEQ ID NO: 3 or SEQ ID NO: 7 which is capable of catalyzing the conversion of sarcosine (N-methyl glycine) to dimethyl glycine and/or the conversion of dimethyl glycine (N,N-dimethyl glycine) to betaine. Preferably, a derivative of a methyltransferase which is capable of catalyzing the conversion of sarcosine (N-methyl glycine) to dimethyl glycine and/or the conversion of dimethyl glycine (N,N-dimethyl glycine) to betaine has about 60 % homology, preferably about 70 % homology, more preferably about 80 % homology, and most preferably about 90 % homology to the corresponding amino acid sequence depicted in SEQ ID NO: 3 or SEQ ID NO: 7. Preferably, when the above amino acid sequences has one or more mutations, substitutions, additions and/or insertions, the amino acids constituting these changes are selected from the 20 standard naturally-occurring amino acids found in proteins, i.e. Ala, Val, Leu, lie, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, Asp, Glu, Lys, Arg and His. Preferably, the mutation (s) and/or substitution (s) are conservative, for example, Ala, Val, Leu, lie, Pro, Phe, Trp, or Met residue (s) are replaced with one of these amino acids, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, residue (s) are replaced with one of these amino acids, Asp or Glu are replaced with one of these amino acids and Lys, Arg or His are replaced with one of these amino acids . In a preferred embodiment, a methyltransferase according to the invention has the amino acid sequence depicted in SEQ ID NO: 3.
In another preferred embodiment, a methyltransferase according to the invention has the amino acid sequence depicted in SEQ ID NO: 7.
The methyltransferase of the present invention capable of catalyzing the conversion of sarcosine (N-methyl glycine) to dimethyl glycine and/or the conversion of dimethyl glycine (N,N-dimethyl glycine) to betaine can exist in the form of an active enzyme or as a zymogen. The term 'zymogen' designates a protein molecule, fragment or derivative thereof (as defined above) which is synthesized in an inactive form and is capable of being activated in vitro or in vivo by the chemical or enzymatic cleavage of one or more peptide bonds . A preferred zymogen of the methyltransferase of the present invention capable of catalyzing the conversion of sarcosine (N-methyl glycine) to dimethyl glycine and/or the conversion of dimethyl glycine (N,N-dimethyl glycine) to betaine comprises the amino acid sequence as depicted in SEQ ID NO: 2 and SEQ ID NO: 3, wherein the N-terminus of SEQ ID NO: 3 is joined to the C-terminus of SEQ ID N0:2.
The methyltransferases of the present invention, for example naturally occurring GSMT and SDMT or recombinantly produced GSMT and SDMT and fragments or derivatives thereof as well as zymogens of these naturally occurring or recombinant proteins, are preferably isolated to a state free from other proteins originating from the organisms from which they are isolated; more preferably, to a pure state, most preferably to a homogeneous state .
Another aspect of the present invention provides an enzyme capable of catalyzing the synthesis of S-adenosyl methionine (SAM), i.e. S-adenosyl methionine synthase (hereinafter referred to as SAMS) which converts methionine to S-adenosyl methionine in the presence of ATP. Preferably, said SAMS comprises an amino acid sequence selected from the group consisting of:
(a) an amino acid sequence as depicted in SEQ ID NO: 4 and an amino acid sequence as depicted in SEQ ID NO: 8,
(b) a fragment of an amino acid sequence as defined in (a) and
(c) a derivative of an amino acid sequence as defined in (a) and (b) .
A fragment of an amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO : 8 is designated as any fragment of an amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO: 8 which is capable of catalyzing the conversion of S-adenosyl methionine from methionine and ATP (adenosine triphosphate) .
A derivative of an amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO : 8 is designated as any mutation, deletion, addition, substitution, insertion or inversion of one or more amino acids, or combination thereof, in the of amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO: 8 which is capable of catalyzing the conversion of S-adenosyl methionine from methionine and ATP. Preferably, a derivative which is capable of catalyzing the conversion of methionine to S- adenosyl methionine has about 60 % homology, preferably about 70 % homology, more preferably about 80 % homology, and most preferably about 90 % homology to the corresponding amino acid sequence depicted in SEQ ID NO: 4 or SEQ ID NO: 8. Preferably, when the above amino acid sequences has one or more mutations, substitutions, additions and/or insertions, the amino acids constituting these changes are selected from the 20 standard naturally-occurring amino acids found in proteins, i.e. Ala, Val, Leu, lie, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, Asp, Glu, Lys, Arg and His. Preferably, the mutation (s) and/or substitution (s) are conservative, for example, Ala, Val, Leu, lie, Pro, Phe, Trp, or Met residue (s) are replaced with one of these amino acids, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, residue (s) are replaced with one of these amino acids, Asp or Glu are replaced with one of these amino acids and Lys, Arg or His are replaced with one of these amino acids .
In a preferred embodiment, the SAMS of the invention has the amino acid sequence depicted in SEQ ID NO: 4.
In another preferred embodiment, the SAMS of the invention has the amino acid sequence depicted in SEQ ID NO: 8.
The SAMS of the present invention, for example naturally occurring SAMS or recombinantly produced SAMS and fragments or derivatives thereof, are preferably isolated to a state free from other proteins originating from the organisms from which they are isolated; more preferably, to a pure state, most preferably to a homogeneous state.
The present invention also relates to a nucleic acid molecule which is capable of encoding a methyltransferase capable of converting glycine to sarcosine (N-methyl glycine) and/or sarcosine to dimethyl glycine (N,N-dimethyl glycine) .
In a preferred embodiment of the present invention, a nucleic acid molecule which encodes a methyltransferase capable of converting glycine to sarcosine (N-methyl glycine) and/or sarcosine to dimethyl glycine (N,N-dimethyl glycine) comprises a nucleotide sequence selected from the group consisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l and a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and (d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a) , (b) and (c) .
A nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO : 1 or a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5 is designated as any nucleotide sequence, for example DNA or RNA, preferably DNA, which hybridizes under standard conditions to a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO: 1 or a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5 and encodes a methyltransferase capable of converting glycine to sarcosine (N-methyl glycine) and/or sarcosine to dimethyl glycine (N,N-dimethyl glycine) . The term 'standard conditions' designates a standard procedure used in heterologous hybridization to screen for genes with enough sequence homology (Maniatis, 1989) . Basically, the hybridization filters are washed first in low stringency conditions and the stringency is gradually increased (usually by increasing the washing temperature) in order to select the positive signals from the background. For example, plaques or colonies of a gene bank to be screened can be transferred onto nitrocellulose membranes and hybridized with a PCR fragment, oligonucleotide or any other cloned DNA containing a fragment of the above gene of interest . The probe can be prepared for example by PCR as described in Example 6. Hybridization can be carried out at 42°C in 50 mM Tris-HCl, pH 7.5 , 10 mM EDTA, 1 M NaCI, 0.5% SDS, 0.1% sodium pyrophosphate, lOx Denhardt ' s solution (Maniatis, 1989) , 100 μg herring sperm DNA and 125 μg/ml polyA. The filters can be first washed with low stringency conditions, for example, at 37°C using 3x SSC, 0.5% SDS and 10% sodium pyrophosphate (Maniatis, 1989) . The filters can then be exposed to x-ray film to monitor the number of positive clones . The washing temperature will be raised in 2-5°C intervals up to a temperature at which the background becomes invisible and only a few positive plaques are obtained. The positive plaques or colonies can then be purified and the DNA can be isolated for Southern blot hybridization to check the size of the cloned insert for example. The cloned DNA obtained can be sequenced. On the basis of the sequence homology, it can be concluded that the DNA contains the gene of interest .
Preferably, a nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO: 1 or a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5 has about 60 %, preferably 70 %, more preferably 80 % and especially 90 % homology to a nucleotide sequence corresponding to a DNA sequence comprising nucleotide 208 to 1047 of SEQ ID NO:l or a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5.
A fragment of a nucleotide sequence from nucleotide 208 to 1047 of SEQ ID NO : 1 or a nucleotide sequence from nucleotide 221 to 1024 of SEQ ID NO : 5 is designated as any nucleic acid fragment, for example DNA or RNA, preferably DNA, which encodes a methyltransferase capable of converting glycine to sarcosine (N-methyl glycine) and/or sarcosine to dimethyl glycine (N,N-dimethyl glycine) .
A preferred nucleic acid molecule according to the invention comprises the DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l.
A further preferred nucleic acid molecule according to the invention comprises the DNA sequence from nucleotide 221 to 1024 of SEQ ID NO : 5
Other preferred nucleic acid molecules according to the invention comprise the DNA sequence from nucleotide 221 to 1867 of SEQ ID NO : 5 or the DNA sequence from nucleotide 208 to 1902 of SEQ ID NO:l, the DNA sequence from nucleotide 221 to 1867 of SEQ ID NO : 5 being more preferred. The present invention also relates to a nucleic acid molecule which encodes a methyltransferase capable of converting sarcosine (N-methyl glycine) to dimethyl glycine and/or dimethyl glycine (N,N-dimethyl glycine) to betaine.
In a preferred embodiment of the present invention, a nucleic acid molecule which encodes a methyltransferase capable of converting sarcosine (N-methyl glycine) to dimethyl glycine and/or dimethyl glycine (N,N-dimethyl glycine) to betaine comprises a nucleotide sequence selected from the group consisting of:
(a) a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l and a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO:5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a) , (b) and (c) .
A nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO: 1 or a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO : 5 is designated as any nucleotide sequence, for example DNA or RNA, preferably DNA, which hybridizes under standard conditions to a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO : 1 or a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO : 5 and encodes a methyltransferase capable of converting sarcosine (N-methyl glycine) to dimethyl glycine and/or dimethyl glycine (N,N-dimethyl glycine) to betaine. The term 'standard conditions' designates a standard procedure used in heterologous hybridization to screen for genes with enough sequence homology (Maniatis, 1989). Basically, the hybridization filters are washed first in low stringency conditions and the stringency is gradually increased (usually by increasing the washing temperature) in order to select the positive signals from the background. For example, plaques or colonies of a gene bank to be screened can be transferred onto nitrocellulose membranes and hybridized with a PCR fragment, oligonucleotide or any other cloned DNA containing a fragment, of the above gene of interest . The probe can be prepared for example by PCR as described in Example 6. Hybridization can be carried out at 42°C in 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1 M NaCI, 0.5% SDS, 0.1% sodium pyrophosphate, lOx Denhardt ' s solution (Maniatis, 1989) , 100 μg herring sperm DNA and 125 μg/ml polyA. The filters can be first washed with low stringency conditions, for example, at 37°C using 3x SSC, 0.5% SDS and 10% sodium pyrophosphate (Maniatis, 1989). The filters can then be exposed to x-ray film to monitor the number of positive clones . The washing temperature will be raised in 2-5°C intervals up to a temperature at which the background becomes invisible and only a few positive plaques are obtained. The positive plaques or colonies can then be purified and the DNA can be isolated for Southern blot hybridization to check the size of the cloned insert for example. The cloned DNA obtained can be sequenced. On the basis of the sequence homology, it can be concluded that the DNA contains the gene of interest .
Preferably, a nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 1048 to 1902 of SEQ ID N0:1 or a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5 has about 60 %, preferably 70 %, more preferably 80 % and especially 90 % homology to a nucleotide sequence corresponding to a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l or a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5.
A fragment of a nucleotide sequence from nucleotide 1048 to 1902 of SEQ ID NO : 1 or a nucleotide sequence from nucleotide 1031 to 1867 of SEQ ID NO : 5 is designated as any nucleic acid fragment, for example DNA or RNA, preferably DNA, which encodes a methyltransferase capable of converting sarcosine (N-methyl glycine) to dimethyl glycine and/or dimethyl glycine (N,N-dimethyl glycine to glycine betaine.
A preferred nucleic acid molecule according to the invention comprises the DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l.
A further preferred nucleic acid molecule according to the invention comprises the DNA sequence from 1031 to 1867 of SEQ ID NO:5.
Other preferred nucleic acid molecules according to the invention comprise the DNA sequence from nucleotide 221 to 1867 of SEQ ID NO : 5 or the DNA sequence from nucleotide 208 to 1902 of SEQ ID NO:l, the DNA sequence from nucleotide 221 to 1867 of SEQ ID NO: 5 being more preferred.
A further aspect of the present invention provides a nucleic acid molecule which encodes an enzyme capable of converting S-adenosyl methionine from methionine and ATP.
In a preferred embodiment of the nuclic acid molecule provided by the present invention, a nucleic acid molecule which encodes an enzyme capable of converting S-adenosyl methionine from methionine and ATP (SAMS) comprises a nucleotide sequence selected from the group consisting of:
(a) a DNA sequence from nucleotide 2027 to 2722 of SEQ ID N0:1 and a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a) , (b) and (c) . A nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO : 1 or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO : 5 is designated as any nucleotide sequence, for example DNA or RNA, preferably DNA, which hybridizes under standard conditions to a DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO : 1 or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO : 5 and encodes enzyme capable of converting S-adenosyl methionine from methionine and ATP. The term ' standard conditions ' designates a standard procedure used in heterologous hybridization to screen for genes with enough sequence homology (Maniatis, 1989) . Basically, the hybridization filters are washed first in low stringency conditions and the stringency is gradually increased (usually by increasing the washing temperature) in order to select the positive signals from the background. For example, plaques or colonies of a gene bank to be screened can be transferred onto nitrocellulose membranes and hybridized with a PCR fragment, oligonucleotide or any other cloned DNA containing a fragment of the above gene of interest . The probe can be prepared for example by PCR as described in Example 6. Hybridization can be carried out at 42 °C in 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1 M NaCI, 0.5% SDS, 0.1% sodium pyrophosphate, lOx Denhardt ' s solution (Maniatis, 1989), 100 μg herring sperm DNA and 125 μg/ml polyA. The filters can be first washed with low stringency conditions, for example, at 37°C using 3x SSC, 0.5% SDS and 10% sodium pyrophosphate (Maniatis, 1989) . The filters can then be exposed to x-ray film to monitor the number of positive clones. The washing temperature will be raised in 2-5°C intervals up to a temperature at which the background becomes invisible and only a few positive plaques are obtained. The positive plaques or colonies can then be purified and the DNA can be isolated for Southern blot hybridization to check the size of the cloned insert for example . The cloned DNA obtained can be sequenced. On the basis of the sequence homology, it can be concluded that the DNA contains the gene of interest . Preferably, a nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 2027 to 2722 of SEQ ID N0:1 or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO: 5 has about 60 %, preferably 70 %, more preferably 80 % and especially 90 % homology to a nucleotide sequence corresponding to a DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO:l or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO: 5.
A fragment of a nucleotide sequence from nucleotide 2027 to 2722 of SEQ ID NO : 1 or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO : 5 is designated as any nucleic acid fragment, for example DNA or RNA, preferably DNA, which encodes enzyme capable of converting S-adenosyl methionine from methionine and ATP.
A preferred nucleic acid molecule according to the invention comprises the DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO:l.
A further preferred nucleic acid molecule according to the invention comprises the DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO : 5.
Further subject matter of the invention is a DNA probe for use in identifying and cloning a nucleic acid molecule encoding a methyltransferase comprising at least 15 nucleotide bases, preferably 20 or more nucleotide bases, of a nucleotide sequence selected from the group consisting of : (a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5. Said DNA probes can be utilized in a method according to the invention for identifying and cloning a nucleic acid molecule encoding a methyltransferase comprising the steps of hybridizing said probe with a sample containing nucleic acid of an organism, detecting a nucleic acid molecule in said sample which hybridizes to said probe and isolating said detected nucleic acid molecule. Preferred methods include the use of the polymerase chain reaction (PCR) and Southern blotting techniques which are described herein and are familiar to the skilled person in the art.
Further subject matter of the invention are vectors for expression of the proteins according to the invention in prokaryotic and eukaryotic hosts.
In this connection, expression vectors, for example phages, plasmids and DNA or RNA viruses, are capable of transforming and/or replicating and expressing the proteins of the present invention in prokaryotes and/or eukaryotes, for example bacteria, yeast, fungi and/or plants. Such expression vectors and methods for their construction are known to the skilled person and can be provided with nucleic acid elements for transcription, for example start codons, 'TATA' boxes, promoters, enhancers, stop codons, etc., and nucleic acid elements important for translation and processing of the nucleic acids transcribed from said vectors in a given host, for example ribosome binding sites, leader sequences for secretion of the proteins of the present invention, etc.
One embodiment of the invention is an expression vector comprising a nucleic acid sequence which encodes a methyltransferase capable of converting glycine to sarcosine (N-methyl glycine) and/or sarcosine to dimethyl glycine (N,N- dimethyl glycine) and/or a nucleic acid sequence which encodes a methyltransferase capable of converting sarcosine (N-methyl glycine) to dimethyl glycine and/or dimethyl glycine (N,N-dimethyl glycine) to betaine. In a preferred embodiment, said expression vector comprises at least one nucleotide sequence selected from the group consisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO: 1 , a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a) , (b) and (c) .
In a further preferred embodiment, an expression vector is provided comprising a nucleotide sequence coding for an enzyme capable of catalyzing the synthesis of S-adenosyl methionine and at least one nucleotide sequence selected from the group consisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and (d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a) , (b)and (c) .
When an expression vector of the present invention contains one of the above mentioned DNA sequences, preferred expression vectors comprise a nucleotide sequence selected from the group consisting of:
(a) the DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, the DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5, the DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO : 1 , or the DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5.
When an expression vector of the present invention contains two of the above mentioned DNA sequences, preferred expression vectors comprise the DNA sequence from nucleotide 208 to 1047 of SEQ ID NO : 1 and the DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO : 1 or the DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5 and the DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5, an expression vector comprising the DNA sequence from nucleotide 221 to 1024 of SEQ ID NO : 5 and the DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO : 5 being more preferred.
However, expression vectors comprising fragments and/or derivatives of the above mentioned sequences as well as other combinations of the above mentioned DNA sequences, for example a DNA sequence from nucleotide 208 to 1047 of SEQ ID N0:1 and a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5 or a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5 and a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l or fragments and/or derivatives thereof are also subject matter of the present invention. As provided for above, expression vectors of the present invention can additionally comprise a nucleic acid molecule coding for an enzyme capable of directly or indirectly increasing the intracellular amount of S-adenosyl methionine.
The genes encoding E. halochloris and A. halophila GSMT and SDMT enzymes are located in a "betaine operon". In E. halochloris the enzymes are encoded by two separate genes, whereas in A. halophila the two enzymes are coded by a single gene. In addition, the "betaine operon" contains a S-adenosyl methionine synthase (SAMS) gene. The SAMS enzyme catalyzes the synthesis of S-adenosyl methionine (SAM) from methionine and ATP, and thus, it is useful in the methylation reactions of the methyltransferases of the invention because it increases the concentration of the enzyme substrate SAM. Therefore co-expression of the SAMS gene with one or more of the methyltransferase genes of the invention can be used to increase betaine synthesis in these organisms.
Hence, in a preferred embodiment, the above mentioned expression vectors can additionally comprise a DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO : 1 or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO : 5 or fragments and/or derivatives thereof when the organism to be transformed is E halochloris or A. halophila.
Preferred expression vectors of this type, which also encode the methyltransferases of the present invention, comprise a DNA sequence from nucleotide 208 to 2722 of SEQ ID NO:l or a DNA sequence from nucleotide 221 to 3004 of SEQ ID NO: 5, the latter being more preferred.
Alternatively, said nucleic acid molecule coding for an enzyme capable of directly or indirectly increasing the intracellular amount of S-adenosyl methionine originates or is derived from the organism which is to be transformed with said expression vector. For example, if the organism to be transformed with a nucleic acid, for example an expression vector, according to the invention is E. coli, then it is possible for example to incorporate the gene coding for S- adenosyl methionine synthase from E. coli (see Markham et al., 1984) . In a similar manner, if the organism to be transformed with a nucleic acid, for example an expression vector, according to the invention is Bacillus, then it is possible for example to incorporate the gene coding for S- adenosyl methionine synthase from Bacillus subtilis (see Yocu et al., 1996, and genebank accession number AF008220) . Other nucleotide sequences which can be used for this purpose are the SAH hydrolase form Mesembryanthemum crystallinum (genebank accession number U79766; Arabidopsis thaliana, accession number AF059581; S. pombe, accession number AL022072) .
In another aspect of the present invention, the expression vectors can additionally comprise a nucleic acid molecule coding for an enzyme capable of increasing the intracellular amount of intracellular glycine. Preferably, said nucleic acid molecule coding for an enzyme capable of directly or indirectly increasing the intracellular amount of glycine originates or is derived from the organism which is to be transformed with said expression vector.
For example, the expression vector can include a nucleotide sequence encoding the enzyme phosphoglycerate dehydrogenase (Bacillus subtilis, accession number L47648; S. pombe, accession number AL022243; Arabidopsis thaliana, accession number AB010407) , phosphoserine aminotransferase (E. coli, accession number AE000193, U00096; Bacillus subtilis, accession number Z99109, AL009126; S. pombe, accession number Z69944; Arabidopsis thaliana, accession number AL031135) , phosphoserine phosphatase (E. coli, accession number AE000509; S. cerevisiae, accession number U36473; S. pombe, accession number D89261) and serine hydroxymethyl transferase (Bacillus thearothermophilius, E02190; Candida albicans, accession number AF009966; Zea mays, accession number W49449) . Further subject matter of the present invention is a recombinant prokaryotic or eukaryotic organism, for example, bacteria, yeast, fungus or plant, transformed with at least one nucleic acid molecule of the invention as defined above, for example, an expression vector according to the invention as defined above.
When a recombinant organism according to the invention is a bacterium, said bacterium is preferably selected from the group consisting of E. coli, Bacillus, Corynebacteria, Pseudomonas and lactic acid bacteria and Streptomyces .
Numerous genetic tools for expressing genes in lactic acid bacteria have been developed in the past few years . The field has extensively been reviewed by uipers et al. (1997) . This opens up many possibilities to develop an inducible expression system for the GSMT or SDMT genes in lactic acid bacteria. In addition, the methodology to express heterologous genes in Bacillus is well established and there are number of functional expression systems facilitating the overexpression of the GSMT and SDMT in Bacillus . (Sarvas, M. (1994) ) .
When the recombinant organism according to the invention is a yeast, said yeast is preferably selected from the group consisting of Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida and Hansenula.
When the recombinant organism according to the invention is a fungus, said fungus is preferably selected from the group consisting of Aspergillus, Trichoderma and Penicillium.
When the recombinant organism according to the invention is a plant including but not limited to cereals, legumes, oilseeds, vegetables, fruits, ornamentals and perennials, said plant is preferably selected from the group consisting of lettuces, Capsicums, grasses, clovers, alfalfa, beans, sweet potatoes, cassava, yams, taro, groundnut, brassica, sugar beet, grapes, potato, tomato, rice, tobacco, rapeseed, maize, sorghum, cotton, soybean, barley, wheat, rye, canola, sunflower, linseed, pea, cucumber, carrot, ornamentals, perennial trees including citrus pear and almond and fruits including strawberry.
In this connection, the term 'plant' according to the invention is understood to include individual cells of a plant, plant seeds and callus material.
Further subject matter of the present invention is a method for the production of a recombinant organism according to the invention comprising the steps of transforming a host prokaryotic or eukaryotic organism, preferably a bacteria, yeast or fungus, with at least one nucleic acid molecule of the invention as defined above, for example an expression vector according to the invention as defined above. When the host organism is to be transformed with a nucleic acid molecule coding for an enzyme capable of increasing the intracellular amount of S-adenosyl methionine and/or glycine which originates from the host organism, this nucleic acid, for example an expression vector, can be transformed as a separate molecule' or can be cloned into a expression vector according to the invention. Likewise, when the host organism is to be transformed with two different nucleic acid molecules each encoding a different methyltransferase of the invention, then transformation can be performed using these two nucleic acid molecules, e.g. expression vectors.
In addition, the present invention relates to a methyltransferase obtainable by culturing wild- ype Ectothiorhodospira or Actinopolyspora or a recombinant prokaryotic or eukaryotic organism according to the invention and isolating said methyltransferase from the organism and/or the medium used to culture or process said organism as well as a method for the production of said methyltransferase comprising the above mentioned steps . In this connection, a method for the purification of a methyltransferase capable of catalyzing the conversion of glycine to dimethyl glycine comprising the steps of subjecting a sample comprising the methyltransferase to a matrix containing adenosine, binding said methyltransferase to said matrix and eluting said methyltransferase from said matrix is also subject matter of the present invention. In addition, the above purification step can be combined with other methods of protein purification including ammonium sulfate precipitation, size exclusion chromatography, cation or anion exchange chromatography, hydrophobic interaction chromatography, etc.
By expressing the genes encoding the GSMT and SDMT enzymes, it is possible to impart the capability of de novo synthesis of betaine to different organisms. Suitable host organisms are practically all bacteria which can be transformed with foreign DNA (for instance E. coli, Bacillus, Corynebacteria, Pseudomonas, lactic acid bacteria and Streptomyces) yeast (for instance Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida and Hansenula) fungi (for instance Trichoderma, Aspergillus, Penicillium) or plants.
Therefore, subject matter of the present invention is a method for the production of betaine comprising the steps of culturing a recombinant organism according to the invention and isolating betaine from the organism and/or the medium used to culture or process said organism.
Further subject matter of the present invention is a method for the production of sarcosine and/or dimethyl glycine comprising the steps of culturing an organism transformed with a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a) , (b) and (c) , and isolating sarcosine and/or dimethyl glycine from said organism or the medium used to culture or process said organism.
Further subject matter of the invention is a method for increasing the intracellular concentration of sarcosine, dimethyl glycine and/or betaine in an organism, enhancing the general productivity of an organism, enhancing the salt tolerance of an organism, enhancing the freezing or cold tolerance of an organism, and/or enhancing the resistance of an organism to drought and/or low water stress comprising the steps of transforming an organism with at least one nucleic acid molecule of the invention as defined above, for example an expression vector according to the invention as defined above. Preferably, said organism is a bacteria, yeast, fungus or plant as recited above .
Further subject matter of the invention is a method for inducing pathogenesis-related proteins in a plant, increasing the resistance of a plant to attack by pathogens and/or increasing the nutritional value of a plant comprising the steps of transforming a plant with at least one nucleic acid molecule of the invention as defined above, for example an expression vector according to the invention as defined above. Preferably, said plant is a plant as recited above.
Pathogens include but are not limited to Fusarium sp. which cases root, shoot and leaf diseases in several plant types, Rhizoctonia εp. and Pythium sp. which cause soil borne diseases in crops, Erysiphe sp. which cause mildew in several species, Phytophthora infestans which causes late blight in potato and tomato, Alternaria solani which causes early blight in potato, fungal diseases of soya caused by Cephalosporium sp., Diaporthe sp., Cerospora sp. Septoria εp. and Peronospora sp., nematodes for example Meloidogne javanica and M. incognita and insects.
Moreover, subject matter of the invention is a method for enhancing the pH tolerance and/or viability of a cultured microorganism comprising the steps of transforming a microorganism with at least one nucleic acid molecule of the invention as defined above, for example an expression vector according to the invention as defined above. Preferably, said microorganism is a bacteria, yeast or fungus as recited above .
The microorganisms of the invention can also be used as hosts in the field of recombinant DNA technology for the expression of a heterologous protein of interest. Therefore, subject matter of the invention is a method for decreasing inclusion body formation, increasing the stability of a heterologous protein and/or increasing the production of a heterologous protein expressed in a microorganism comprising the stepε of transforming a microorganism with at least one nucleic acid molecule of the invention as defined above, for example an expresεion vector according to the invention aε defined above, and tranεforming a microorganism with a nucleic acid molecule capable of expresεing εaid heterologouε protein. Said microorganism can be transformed with the nucleic acid molecule, for example an expression vector, according to the invention before, during or after the microorganism is transformed with a nucleic acid molecule capable of expressing said heterologous protein.
Additional subject matter of the invention iε an animal feed or animal feed ingredient compriεing a recombinant organism according to the invention. The present invention is more closely illustrated by means of the following examples without limiting the invention to the examples .
Examples
Example 1. Demonstration of the methyltransferase pathway in Actinopolyspora halophila and Ectothiorhodospira halochloris
Preparation of the cell extracts
The growth medium of Actinopolyspora halophila ATCC 27976 used in all cultivations was the "complex medium" described by Sehgal and Gibbons (1960) . Inoculum was grown at 37°C in a shake flask with agitation at 180 rpm until the late exponential growth phase. Then, 8 1 of the above medium with 10 g/l glucose was inoculated with 800 ml culture. The pH in the fermentor (Biostat M (Braun) laboratory fermentor) waε maintained at pH 6.5-7.5 with 0.5 M H2S04 and 1 M NaOH. Agitation and aeration rates were 400 rpm and 10 1/min., respectively. The cultivation temperature was 37°C. Cells were grown to late exponential phase and harveεted by centrifugation at 15,000 g for 15 min. Cellε were stored at -75°C. Before disruption, the cells were thawed and suspended in Buffer I (22 % (w/v) sucrose, 27 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF) , 50 mM Tris-HCl, pH 7.5) in a ratio of 1.5 ml buffer 1:1 g cells (wet weight) .
The growth medium used in the cultivation of Ectothiorhodoεpira halochloriε ATCC 35916 is described by Tschichholz and Trύper (1990) . Cultivation was carried out anaerobically at 42°C in 1 1 glasε bottleε with continuouε εtirring with magnetic stirrer. The cellε were illuminated during growth (5,000 - 10,000 lux). 100 ml of pre-inoculum waε inoculated into 1 1 medium. Cellε were grown until late exponential phaεe and harveεted by centrifugation at 28,000 g for 20 min. After centrifugation, the cells were suεpended in Buffer II (560 mM Tris-HCl, pH 7.5, 4 mM 2-mercaptoethanol, 50 μM MgCl2, 160 μM EDTA) and disrupted with 1 mM PMSF and 1 mM dithiothreitol (DTT) . Buffer II was added in a ratio of 1.5 ml buffer II: 1 g cells (wet weight) .
The cells were disrupted with a MSE Soniprep 150 sonicator. The suspension of A. halophila cells waε εonicated in 20 ml batches (sonication pulses 30 s, cooling intervals 2 min) for 1 min / 2 ml cell suspension. The suspension of E. halochloris cells was sonicated in 5 ml batches (sonication pulseε 15 s; cooling intervals 2 min) for 1 min/1.5 ml cell suεpension. The cell debris was removed by centrifugation at 28,000 g at 1°C for 30 min. The cell free extracts were stored at -75°C.
Methyltransferase activity asεay
Reactions were carried out in 1.5 ml Eppendorf tubeε with capε . The reaction mixture contained 25 μl of 0.1 M substrate (glycine, sarcosine or dimethylglycine) , 25 μl of Buffer II (see above) , 25 μl 4 mM S-adenosyl-L-methionine containing 45 nCi S-adenosyl-L- [methyl-14C] methionine (Amersham) in 1/10 Mcllvaine buffer (pH 3.0), and 25 μl enzyme sample (e.g. cell free extract) . The reaction was initiated by adding the enzyme. The reaction mixture was incubated for 30 min. at 37°C and the reaction was stopped by adding 75 μl of charcoal suspension (133 g/1 in 0.1 M acetic acid) . The excesε charcoal selectively adsorbε unreacted S-adenosyl methionine. The reaction mixtures were then incubated for 10 min at 0°C and centrifuged for 10 min in a Heraeus table top centrifuge. 75 μl of the supernatant was added to 4.5 ml of aqueous scintillant (Hionic-Fluor, Packard) and the radioactive methylation productε were meaεured in a liquid scintillation counter (Beckman LS 6000 IC) . The enzyme sample was diluted to keep the reaction on the linear range (radioactivity of the supernatant below 10,000 DPM) .
The cell extracts typically contain the following activities. Table 1. Methyltranεferaεe activitieε of A. halophila and E. halochloris cell extracts on different substrates.
* The activity is expressed in units based on the radioactivity of the reaction products after 30 min. incubation
Characterization of the methylation reaction products in the reaction mixtures
The reaction products were characterized by HPLC. The reaction mixture supernatants aε deεcribed above were filtered after centrifugation through a Miniεart NML 0.2 μm filter (Sartorius AG) and a 25 - 100 μl sample was analyzed on AminexHPX-87C cation exchange column (300 x 7.8 mm) (BioRad Laboratories) . The HPLC system used was a Varian 500 equipped with a HP 1047 (B) efractive index detector and a Waterε VISP717 injector. A μbondapack C18-precolumn was uεed in the εyεtem. 5 mM CaS04 was used as the eluent and the flow rate was 0.6 ml/min. A 1 mM mixture of sarcoεine, dimethylglycine and betaine were used as standards . In order to detect the radioactive products formed in the enzymatic reaction, 200 μl fractions were collected during the chromatographic run. The fractions were analyzed in a liquid scintillation counter as described above. The dead-volume between the detector and εample outlet was determined by radioactive betaine and the retention time of the different fractions was calculated from the sample volume and eluent flow rate. The radioactivity of the fractionε waε plotted to the εame Figure with the εtandardε, which were uεed to identify the reaction products. The results obtained by using A. halophila cell extracts are presented in Figure 1. The resultε indicate that all methylation products in the three-step reaction are formed during the incubation.
Example 2. Purification of glycine-sarcosine methyltransferase (GSMT) of E. halochloris
20 mM Tris-HCl buffer (pH 7.5) was used through the purification procedure unless otherwise stated. All the bufferε used contained 1 mM dithiothreitol .
Step 1: Ammonium sulphate fractionation. 25 ml of cell free extract (as described in example 1) was diluted to 90 ml and saturated ammonium sulphate in 50 mM Tris-HCl, pH 7.5, was added to achieve 20 % saturation. The solution was incubated for 30 min at 0°C and centrifuged at 15,000 g. The precipitate was discarded and the supernatant purified further.
Step 2. Hydrophobic interaction chromatography. The supernatant from step 1 (105 ml) was applied to a Butyl Sepharose 4 FF (Pharmacia) (10 x 50 mm) column pre-equilibrated with 20 % (w/v) ammonium sulphate in 20 mM Tris-HCl, pH 7.5. The column was washed with 45 ml of 20% (w/v) ammonium sulphate in 20 mM Tris-HCl and eluted with a linear gradient of 20-0% ammonium sulphate. The volume of the gradient waε 80 ml and the flow rate was 2 ml/ in. Fractions of 3 ml were collected. The active fractions (40 ml) were pooled. The ammonium sulphate was removed by gel filtration (Sephadex G-2S, Pharmacia) .
Step 3. Ion exchange chromatography. The sample from step 2 (73 ml) was applied to a DEAE-Memsep 1000 HP (Millipore) (1.4 ml) column pre-equilibrated with 20 mM Tris-HCl, pH 7.5. The column was washed with 15 ml of buffer and eluted with a linear NaCI gradient (0 - 1 M) . The volume of the gradient was 60 ml and the flow rate was 3 ml/min. 2 ml fractions were collected The active fractionε (8 ml) were pooled and concentrated by ultrafiltration (A icon Centriplus 30; Ultrafree MC 10,000 NMWL filter unit Millipore) to 100 μl.
Step 4. Gel filtration. The concentrated sample from step 3 (100 μl) was applied to a Superose 12 HR 30 (Pharmacia) column 20 mM Tris-HCl, pH 7.5, containing 150 mM NaCI was uεed aε the elution buffer with flow rate of 0.4 ml/min. 0.5 ml fractionε were collected. The fractionε containing glycine-sarcosine methyltransferase (GSMT) activity (1.5 ml) were collected and concentrated by ultrafiltration (Ultrafree MC 10,000 NWML filter unit, Millipore) to 100 μl .
The purity and the molecular weight were determined by gradient SDS-polyacrylamide gel electrophoresis according to the following procedure. Electrophoresis under denaturing conditions was carried out using pre-made polyacrylamide gel εlabε (12 % Triε-glycine gel with 4 % stacking gel, Ready Gelε, Biorad) according to the inεtructions of the manufacturer. Mid range molecular weight standard from Promega was used. Staining of the gel was performed with 0.25% (w/v) Coomassie Blue R-250 (Promega) in 50 % (v/v) methanol and 10% (v/v) acetic acid. Stained gels were deεtained with 10% (v/v) methanol and 5% acetic acid (Laemmli, 1970) .
A typical SDS-gel of the purified E. halochloris GSMT is shown in Figure 2.
Example 3. Purification of sarcosine-dimethylglycine methyltransferase (SDMT) of Actinopolyspora halophila
An affinity purification method waε developed for the purification of the sarcosine-dimethylglycine methyltransferaεe (SDMT) . Purification of the protein was not achieved by standard purification methodology. The affinity column was prepared as follows. 5 'AMP-Sepharose 4B (Pharmacia) was treated with alkaline phosphataεe to remove the phoεphate group of the ligand. The gel waε firεt εwollen in water (5 ml of distilled water was uεed per 1 g of dry 5 'AMP-Sepharoεe 4B) . The εwollen gel waε then waεhed with 200 ml of diεtilled water. The gel waε equilibrated with CIP-buffer (10 mM MgCl2, 1 mM dithiothreitol, 50 mM NaCI, 10 mM Triε-HCl, pH 7.9). 10 μl (100 U) of CIP (Calf Intestinal Alkaline Phosphatase (Finnzymes) ) was added to the gel. The gel was incubated for 2 h at 37°C with occaεional shaking. The reaction was stopped by washing the gel with 20 mM Tris-HCl, pH 7.5.
18 ml of A. halophila cell free extract (as described in example 1 ) waε applied to a column of adenoεine-Sepharose (10 x 90 mm) pre-equilibrated with 20 mM Tris-HCl, pH 7.5. The column waε waεhed with 20 mM Triε-HCl, pH 7.5, uεing 0.5 ml/min flow rate until the absorbence at 280 nm became constant. The protein bound to the column waε eluted with 1 mM S-adenoεyl methionine (20 mM Triε-HCl, pH 7.5). The active fractionε were pooled (25.2 ml) and concentrated by ultrafiltration (Amicon Centripluε 30) . The purity and the molecular weight was determined by gradient SDS-polyacrylamide gel electrophoresis as described above.
A typical SDS gel of the purified A. halophila SDMT is shown in Figure 2.
Example 4. Characterization of the properties of A. halophila SDMT protein
Determination of the molecular weight and sub-unit structure
Based on the SDS-electrophoresis (Figure 2) , the molecular weight of A. halophila SDMT is approximately 32 kDa.
The molecular weight was also determined by gel filtration with Superose 12 HR 30 (Pharmacia) column. The flow rate waε 0.4 ml/ml. The elution buffer was 20 mM Tris-HCl pH 7.5 containing 150 mM NaCI.
The molecular weight was calculated from a calibration curve made with a mixture of standard proteins . The mixture contained 0.5 mg/ml Blue Dextran (0.5 mg/ml), Ferritin (440 kDa), 7.0 mg/ml aldolase (158 kDa), 2.0 mg/ml ovalbumin (43 kDa) and 1.0 mg/ml chymotrypsinogen (25 kDa). The calculated molecular weight waε 31.6 kDa, which indicates that the protein is a monomer.
The isoelectric point of the enzyme
The isoelectric focusing waε performed with Pharmacia Phast system using gels with pH-gradient from pH 3 to 9 (IEF 3-9) . A mixture of Pharmacia IEF standard proteins with pis from 3.5 to 9.3 were uεed aε standards. The gels were stained by silver staining as described in example 1. The resultε shown in Figure 3 show that the pi of the protein is approximately 4.1-4.2.
Substrate specificity
The activity of the purified protein was determined as described in example 1 with glycine, sarcosine and dimethyl glycine. The data presented in table 2 demonstrates that the isolated protein catalyzes step methylation reaction from sarcosine to dimethyl glycine and from dimethyl glycine to betaine.
Table 2. The activity of A. halophila SDMT on different subεtrateε .
* The activity iε expressed in arbitrary units based on the radioactivity of the reaction products after 30 min. incubation.
pH Optimum
The pH-optimum of the two methylation reactionε were determined by uεing following buffers: 0.1 M piperazine buffer, pH 5.0; 0.1 M Bis-Triε buffer, pH 6.0; 0.1 M Bis-Tris buffer, pH 7.0; 0.1 M Tris-HCl, pH 8.0; 0.1 M Tris-HCl, pH 9.0; 0.1 methanolamine, pH 10. The exact pH valueε of the reaction mixtureε were measured. It can be concluded from Figure 4 that the pH optimum of the both enzyme reactions is at pH 7.5.
Temperature optimum
The temperature dependence of the enzymatic reactionε were determined with sarcosine and dimethyl glycine. As seen in Figure 5. the temperature optimum is approximately 45 - 50°C. When the temperature is elevated above 50°C, the enzyme is rapidly inactivated.
Example 5 In vitro synthesis of betaine by using purified E. halochloris GSMT and A. halophila SDMT
The purified GSMT from E. halochloriε and SDMT from A. halophila were concentrated by ultrafiltration (Ultrafree MC 10,000 NWML filter unit, Millipore) to protein concentrations 4.2 mg/ml and 5.6 mg/ml, respectively. The protein concentration was determined by measuring the absorbance at 280 nm and calculated by the formula: 1 mg protein/ml = 1.0
A280-
The reaction mixture contained 50 μl 5.0 mM glycine, 50 μl 32 mM S-adenosyl methionine in water containing 640 nCi S-adenosyl-L- [methyl-1 C] -methionine (Amersham) , 50 μl Buffer II (see example 1), 25 μl GSMT of E. halochloris and 25 μl SDMT of A. halophila. The reaction was initiated by adding the enzymes . The reaction mixture waε incubated for 2 h at 37°C and the reaction was stopped by adding 150 μl of charcoal suspension. The reaction mixtures were then incubated for 10 min at 0°C and centrifuged for 10 min in a Heraeuε table top centrifuge. The εupernatantε were filtered through Miniεart NML 0.2 μm filter (Sartorius AG) . The identification of the reaction products was performed by HPLC as described in example 2.
The chromatogram is presented in Figure 6 and it showε peakε corresponding to the retention times of sarcosine, dimethylglycine and betaine.
Example 6. Isolation of the genes of E. halochloris GSMT and A. halophila SDMT
Determination of the N-terminal and internal peptide sequenceε
The N-terminal and tryptic peptideε peptide εequences of the purified proteins were determined by using Perkin Elmer/Applied Biosyεtems Procise 494A protein εequencing system as described by Kerovuo et al . , 1998. The peptide εequences obtained are shown in table 3.
Table 3. The peptide sequenceε obtained from purified E. halochloriε GSMT and halophila SDMT. The εequenceε uεed to make the PCR primerε are underlined.
Organism/SEQ ID NO: Sequence
A. halophila
SEQ ID NO: 9 EKSYRTEDEFVDMYSNAVHTARDYYNSEDASNFYYHV
(N-terminus) SEQ ID NO-.10 GSVLFTDPMASDDAK SEQ ID NO: 11 TGLRNYQAGN SEQ ID NO: 12 LXELGPILDRLHLDSG
SEQ ID NO: 13 ELTRLGLONIEFEDLSEYLPVHYGR
SEQ ID NO: 14 VDISPETRILDLGSGYGA E. halochloris.
SEQ ID NO: 15 NTTT/EEODFGADPTKVRDTDAYTE
(N-terminus)
SEQ ID NO: 16 VRDTDHYTEEYVD
SEQ ID NO: 17 DYTRRLMHEVGFQK
SEQ ID NO: 18 ATYRDADPDFFLHVAEK
SEQ ID NO: 19 VRDTDHYTEEYVDGFVDKWDDLID
Preparation and screening of the chromosomal gene banks
The genomic DNA from both microbes was isolated essentially as described in Ausubel et al . (1991). The chromoεomal DNAs were partially digested with Sad and ligated to Sad digested dephosphorylated lambda ZapII arms (Stratagene, La Jolla, California, USA) and packaged to lambda particleε uεing Gigapack III Gold packing extract (Stratagene, La Jolla California, USA) according to protocol provided by manufacturer. The chromoεomal DNA isolated from the organisms was used as the template DNA in the PCR reactions .
The probes were made by PCR using following degenerate primers. The primers were designed according to Sambrook et al. (1989) .
A. halophila
SEQ ID NO: 20
5 ' -GA (A/G) GA (C/T) GA (A/G) TT (C/T) GTIGA (C/T) ATG T-3 '
SEQ ID NO: 21
(5 ' - (C/T) TG (A/G) TT (T/A/G) AT (T/C) TC (G/A) AA (T/C) TC (A/G) TC-3 ' )
E. halochloris
SEQ ID NO -.22
5 ' -GA (A/G) CA (A/G) GA (T/C) TT (T/C) GGIGCIGA (T/C) CC-3 ' SEQ ID NO: 23
5 ' -A(A/G) (A/G) AA (A/G) AA (A/G) TCIGG (A/G) TCIGC (A/G) TC-3 '
The amplification waε performed under the following conditions.
A. halophila- 3 cycles of 1 min. at 94°C for denaturation, 1 min at 37°C annealing and 2 min at 72°C for εynthesis, 32 cycleε of 1 min at 94° C for denaturation, 1 min at 46°C annealing and 2 min at 72°C for synthesis. AmpliTac DNA Polymerase (Perkin Elmer) waε used in the reaction. 1 mM MgCl2 was added to the reaction mixture. Otherwise standard reagents were used.
E. halochloris - 34 cycles of 1 min at 94° for denaturation, 1 min at 42°C annealing and 2 min at 72 °C for syntheεiε. AmpliTac DNA Polymeraεe (Perkin Elmer) waε uεed in the reaction. 1 % (v/v) formamide, 1% (v/v) dimethyl sulfoxide and 6 mM MgCl2 were added to the reaction mixture. Gitsch-buffer (reference) was used in the reaction.
The PCR-fragments obtained were labeled with rediprime DNA labelling syεtem (Amersham Life Science) according to the instructionε given by the manufacturer.
A total of 50,000 plaqueε of both libraries were screened and the positive lambda clones were cored and excised with ExAssiεt helper phage (Stratagene, San Diego, USA) to obtain phagemids. CsCl-gradient purified (Sambrook et al., 1989) plaεmid DNAε were used in DNA sequencing (Zagursky et al., 1986). The lengths of the cloned fragments were 3.5 kb (A. halophila) and 5.0 kb (E. halochloris).
Analyεiε of the εequencing data
E. halochloris - Sequence analysis of the DNA fragments indicated that the E. halochloris clone contains 3 ORFs . On the basis of the peptide sequences it can be concluded that the E. halochloris GSMT is encoded by the first ORF of the fragment. In addition, based on the sequence homology with the A. halophila SDMT, it can be concluded that the second ORF of E. halochloris clone contains a SDMT gene. This has also been demonstrated by expresεing the gene in E. coli (Example 8) .
A. halophila - Sequence analysis of the DNA fragments indicated that the A. halophila clone contains 2 ORFs . A. halophila SDMT protein is coded by the 3 '-end of the first ORF of the A. halophila clone. The 5 '-end of the same ORF is very homologous to the E. halochloris GSMT. These data indicate that the GSMT and SDMT are transcribed from a single gene and the protein isolated from the organism is a procesεing product.
In addition to the methyltranεferaεeε, the "betaine operon" codes for a third gene which is homologous to number of S-adenosyl-methionine synthases. The operon εtructure is schematically shown in Figure 7. The nucleotide and amino acids sequences of the cloned genes have been shown in Figures 8 and 9.
Example 7 Expresεion of E. halochloris GSMT in E. coli
Expression of the gene
The gene coding for the E. halochloris GSMT was amplified by PCR. The purified plasmid used for DNA sequencing in example 6 was used as the template for the PCR reaction. The following primers were used in the PCR reaction:
primer 1 :
5 ' -CGGACCATGGATACGACTACTGAGCAG-3 ' (SEQ ID NO : 24)
(5 '-end oligonucleotide) and primer 2 :
5' -GCTCAGATCTGTCCTCCTCCCGATATTCCTTCTC-3' (SEQ ID NO: 25)
(3 ' -end oligonucleotide)
The 3 '-end of the primers are homologous to the 5'- and 3 ' -end of the GSMT gene. The 5 '-end oligonucleotide hybridizes to position 221-241 and the 3 '-end to the position 1001-1024. (See Figure 8) . The primer hybridizing to the 5'- end contains an extra Ncol restriction site such that the nucleotide A at position 224 in Figure 8 iε replaced by the nucleotide G in the primer and the 3 '-end primer containε a Bglll site which were used for cloning.
The amplification was performed in the following conditions : 34 cycles of 1 min at 94°C for denaturation. 1 min at 50 °C annealing and 2 min at 72°C for syntheεiε. Pfu Polymeraεe
(Stratagene) waε uεed in the reaction. The amplified fragmentε were purified with Qiaquick DNA purification Kit
(Qiagen, Santa Clara, USA) and ligated into Ncol/Bglll cut PQE-60 expreεεion vectorε (Qiagen, Santa Clara, USA) . A εchematic preεentation of the plaεmid (pEGSM) is shown in Figure 10. Competent XL-1 Blue MPF1 cells were transformed with this ligation mix according to Hanahan et al . (1983) . Plasmid minipreps of the tranεformantε were prepared and the presence of the inεert waε eεtablished by cutting the plasmids with Nco I and Bgl II and by separating the resulting fragments by agarose gel electrophoresis.
GSMT transformants were grown overnight in 2.5 ml of LB broth containing 100 μg/ml ampicillin. As a control, E. coli XLI
Blue MRF' transformed with the PQE-60 without the insert was grown.
0.5 ml was inoculated to 1.5 ml of LB broth with ampicillin. The cultures were grown at 200 rpm for 30 min at 37°C and isopropyl-β-thiogalactopyranoside (IPTG) waε added to a final concentration of 1 mM to induce the enzyme εyntheεis. After 3 h 30 min the cellε were separated by centrifugation (1,000 g, 3 min) .
The cell pellet was suεpended in 100 μl Buffer II containing 1 mM PMSF (See example 1) and the cellε were diεrupted with a MSE Soniprep 150 εonicator. The cell suspension was εonicated with εonication pulses of 5 ε for 10 s . The sampleε were cooled on ice between the pulses . The cell debris was removed by centrifugation at 13,000 rpm for 30 min at 4°C in a Heraeus table top centrifuge. The activitieε of the supernatants were determined aε in example 1.
The activity of the cell extractε of the tranεformantε waε assayed as described in example 1. The activities using glycine and sarcoεine aε substrates were typically 3,000-5,000 dpm/30 min and 1,000-2,000 dpm/30 min., respectively.
Salt tolerance of E. coli clones expressing the E. halochloris GSMT
The strainε uεed in theεe teεtε were the poεitive clone deεignated EGSM and E. coli XLI Blue MRF' tranεformed with the cloning vector PQE-60. The growth medium uεed in this test was the synthetic medium MM63 deεcribed by Larεen et al . (1987) supplemented with 1.5 mil/1 of vitamin solution VA (Imhoff and Trύper, 1977) and 100 μl/ml ampicillin.
The bacterial strainε were grown to mid-exponential growth phases with shaking at 180 rpm at 37°C and centrifuged (1,000 g, 15 min) . The cells were resuεpended in the growth medium to abεorbance of 0.9 at 600 nm.
The automatic turbidimetric εyεtem Bioscreen (Labsystems) waε uεed for monitoring the growth. 30 μl waε inoculated to 300 μl of the medium with 0 or 0.2 M NaCI and 1 mM IPTG (final concentrations) . Cells were grown at 37°C with intensive and continuous shaking. Growth was followed by absorbance measurements in Bioscreen at 600 nm.
The growth curves of EGSM and E. coli transformed with PQE-60 in a medium without added NaCI are presented in and with 0.2 M NaCI in Figure 11. It can be concluded from the data that EGSM has an increased tolerance towards oεmotic stress.
Example 8. Expression of E. halochloris SDMT in E. coli
The gene encoding the E. halochloris SDMT was amplified by PCR. The purified plasmid used for DNA sequencing in example 6 was used aε the template for the PCR reaction. The following primerε were used in the PCR reaction:
primer 3 :
5 ' -GCATGCCATGGCGACGCGCTACGACGATCAA-3 ' (SEQ ID NO : 26)
(5 -end oligonucleotide) and
primer 4 :
5 ' -GGGAAGATCTCCCTTTGCGGAAGTAAAAGATACC-3' (SEQ ID NO:27)
(3' -end oligonucleotide)
The primers are homologous to the 5'- and 3 ' -end of the E. halochloris SDMT gene. The 5' -end oligonucleotide hybridizes to position 1031-1054 and the 3 '-end to the position 1844- 1867 (Figure 8) . The primer hybridizing to the 5' -end contains an extra Ncol restriction site and the 3 '-end primer a Bglll site which were used for cloning of the fragment.
The preparation of the DNA construct and the cultivation of the poεitive E. coli clones were done according to example 7. A schematic presentation of the expression plasmid (pESDM) is shown in Figure 10.
The cultivations and preparation of the cell-free extracts were performed essentially as described in example 7. The εonication of the cell extract pulses was shortened to 3 x 2 second intervalε (total sonication time was 6 s).
The activity of the cell extracts of the transformantε waε aεsayed as described in example 1. The activities using sarcosine and dimethyl glycine as substrateε were typically 20,000 dpm/30 min. with both εubεtrateε .
Example 9. Co-expression of E. halochloris GSMT and SDMT in E. coli
The DNA construct made for this experiment containε both GSMT and SDMT genes separated by a short (3 nucleotides long) linker. The DNA fragment was obtained by amplification of the purified plaεmid used for DNA sequencing in example 6.
The following primers were used in the amplification:
primer 1 :
5 ' -CGGACCATGGATACGACTACTGAGCAG-3 ' (SEQ ID NO :24)
(5 '-end oligonucleotide) and
primer 4 :
5 ' -GGGAAGATCTCCCTTTGCGGAAGTAAAAGATACC-3' (SEQ ID NO:27)
(3 ' -end oligonucleotide)
The primers are homologous to the 5 '-end of the E. halochloris GSMT and the 3 ' -end of the E. halochloris SDMT gene. The 5 '-end oligonucleotide hybridizes to position 221- 241 and the 3 ' -end to the position 1844-1867 (See Figure 8) . The primer hybridizing to the 5 '-end contains an extra Ncol restriction site and the 3' -end primer a Bglll site.
The preparation of the DNA construct and the cultivation of the positive E. coli clones were done according to example 7. A schematic presentation of the expreεεion plaεmid (pEhFU) iε shown in Figure 10. The induction and preparation of the cell-free extracts was performed esεentially as described in example 9
The enzymatic activities were asεayed as described in example I . The cell extracts of the transformantε clearly εhowed activity with glycine, εarcosine and dimethylglycine. The activities of the cell extracts with the three substrateε were all over 20,000 dpm/30 min.
Salt tolerance E. coli cloneε expreεεing the E. halochloriε GSMT and SDMT
The test was performed esεentially aε described in example 7. The poεitive clone deεignated EhFU was used in this teεt.
The growth curves of EhFU and E. coli with PQE-60 in a medium without added NaCI are presented in and with 0.2 M NaCI in Figure 12. It can be concluded from the data that EhFU has an increased tolerance to osmotic stress.
Betaine synthesiε in E. coli clones expresεing the E. halochloriε GSMT and SDMT
The growth medium used in this test was the synthetic medium MM63 described by Larsen et al. (1987) εupplemented with 1.5 ml/1 of vitamin solution VA (Imboff and Trύper, 1977) and 100 μl/ml ampicillin. The medium contained 1% (wlv) glucose.
The clone EhFU (Figure 10) and the control strain (E coli XLl-Blue MRF' transformed with the cloning vector PQE-60) were grown to mid-exponential phaεe with εhaking at 180 rpm at 37°C. The cellε were centrifuged at 1,000 g for 10 min and resuspended in the growth medium εo that the turbidity goot was 0.640. 5 ml of this cell suspension waε inoculated to 50 ml of media containing 0.22 or 0.33 M NaCI and 25 mM L-methionine. The bacterial εtrainε were grown for 2 h with εhaking at 180 rpm at 37°C and 1 mM IPTG was added. Growth was followed by measuring the turbidity at 600 nm. The cells were grown to early εtationary phaεe. Cellε from 45 ml of culture were harvested by centrifugation at 1,000 g for 15 min and washed once with the growth medium without glucose.
The cell pellets were suspended in 2 ml of water and kept in a boiling water bath for 10 min. The suεpenεion was centrifuged for 15 min at 23,000 g and the supernatant collected and the pellet resuspended in water. This extraction was repeated twice. The three supernatants were combined. The volumes of supernatantε were measured and the supernatantε were filtered and analyzed by HPLC as described in example 1.
A similar experiment was performed also without added L-methionine.
The betaine produced inside the cells is presented in table 4.
Table 4. The amount of betaine syntheεized inside the cells in 1 ml of culture when grown in MM63
The resultε εhow that the E. coli clone expressing the E. halochloris GSMT and SDMT genes εyntheεizeε betaine in theεe cells. The highest amount of betaine synthesized corresponds roughly to 1% (0.2 M NaCI) and 0.5 % (0.3 M NaCI) of the cell dry weight
Example 10. Expression of the DNA fragment encoding the protein isolated as A. halophila SDMT in E. coli
The gene sequencing results revealed that a single gene codes for the A halophila GSMT and SDMT. The fusion protein was not, however, succesεfully purified from the A. halophila cell extractε . Instead a protein with SDMT activity was isolated. In this experiment the corresponding part of the GSMT-SDMT gene iε expresεed in E. coli.
The gene fragment encoding the SDMT enzyme activity was amplified by PCR. The genomic DNA from A. halophila isolated in example 6 was used as the template for the PCR reaction. The following primers were used in the amplification:
primer 5 :
5 ' -GCTGCCATGGAGAAGAGCTACCGCACCGAG-3' (SEQ ID NO:28)
(5'-end oligonucleotide) and
primer 6 :
5'-GGGAAGATCTTGCCCTGGCGTGGATGATGCCCCA-3' (SEQ ID NO:29)
(3-end oligonucleotide)
The primers are homologous to the 5'- and 3 '-end of the ASDMT gene. The 5' -end oligonucleotide hybridizes to position 1048- 1068 and the 3' -end to the position 1879-1902 (See Figure 9) . The primer hybridizing to the 5 '-end contains an extra Ncol restriction site and the 3 '-end primer a Bglll site.
The preparation of the DNA construct and the cultivation of the positive E. coli cloneε were done according to example 7. A εchematic preεentation of the expression plasmid (pASDM) is shown in Figure 10. The induction and preparation of the cell free extracts was performed essentially aε deεcribed in example 9. The enzymatic activities were assayed as described in example 1. The cell extracts of the transformants clearly showed SDMT activity. The activities on sarcosine and dimethyl glycine were typically 20,000 dpm/30 min. with both substrates. There was no activity on glycine.
Example 11. Expression of A. halophila GSMT-SDMT fusion protein in E. coli
The gene fragment encoding GSMT and SDMT of A. halophila was amplified by PCR. Primers uεed were:
primer 7 :
5 ' -CATGCCATGGCCAAGAGCGTGGACGATCTT-3 ' (SEQ ID NO : 30)
(5 '-end oligonucleotide) and
primer 6 :
5 ' -GGGAAGATCTTGCCCTGGCGTGGATGATGCCCCA-3 ' (SEQ ID NO : 29)
(3-end oligonucleotide)
The primerε are homologous to the 5'- and 3 ' -end of the AGSMT-ASDMT gene. The 5 '-end oligonucleotide hybridizes to position 208-231 and the 3 '-end to the position 1879-1902 (See Figure 9). The primer hybridizing to the 5 '-end contains an extra Ncol restriction site εuch that the nucleotide A at poεition 211 in Figure 9 iε replaced by the nucleotide G in the primer and the 3 '-end primer containε a Bglll εite.
The purified plaεmid used for DNA-sequencing in example 6 was used as a template for the PCR reaction. The amplification was performed in following conditions: 34 cycles of 30 s at 94°C for denaturation, 1 min at 50°C annealing and 2 min at 72°C for εynthesis. Ligation of the amplification product into Ncol/Bglll cut PQE-60 and the transformation of XL-1 Blue MRF' cells was performed as in example 7. A εchematic presentation of the expresεion plaεmid iε shown in Figure 10. The induction and preparation of the cell-free extracts was performed essentially as in example 9 except that the sonication pulses were shortened to 2 ε and the total εonication time to 6 s . The cell-free extract was analyzed by SDS-polyacrylamide gel electrophoresis as in example 2. The pellet from the centrifuged suspension waε suspended to 10 mM Triε-HCl-buffer, pH 8.0 containing 8 M urea and 0.1 M Na3P04 to εolubilize the proteins of the pellet and centrifuged for 15 min in a Heraeus table top centrifuge at 13,000 rpm. The εupernatants were analyzed by SDS-polyacrylamide gel electrophoresis as in example 2.
The enzymatic activities were assayed as described in example 1. The cell extracts of the transformantε clearly showed SDMT activity. The activities on sarcosine and dimethyl glycine were typically 10,000 dpm/30 min and 20,000 dpm/30 min., respectively. There was no activity on glycine.
The SDS-polyacrylamide gel of the cell-free extract showed no major protein band of correct εize. However, the insoluble pellet solubilized with 8 M urea showed a major band corresponding to the molecular weight of the GSMT-SDMT fusion protein. The results indicate that when A. halophila GSMT- SDMT is over-expresεed in E. coli it forms inclusion bodies. However, a fraction of the protein - which corresponds the SDMT - is proteolytically cleaved and remains soluble in the cells .
Example 12. Expression of E. halochloris GSMT and SDMT in tobacco and potato
Tobacco and potato plants can be transformed by AgrroJacteriu-n mediated tranεformation system. Identical DNA construct can be used for both plants.
The GSMT gene is first tranεformed into the plant uεing a plaεmid containing a kanamycin reεistance marker. Positive transgenic plants obtained by screening for the enzyme activity are then used as host plants for second transformation of the SDMT gene. Another εelection marker, hygromycin selection is used in the second transformation. Experiments are performed using stable transformants of the Fx generation.
The genes of E. halochloris GSMT and SDMT are amplified by PCR by using plasmid pEFU (see example 10) as the template. The primers used hybridize to the same regions of the DNA as shown in Fig. 8 (GSMT: primer 1 and primer 2; SDMT; primer 3 and primer 4) . The final DNA constructs are made using suitable restriction sites to transfer the genes to plant transformation vectors. PBin 19 based pGPTV vectors (Becker et al, 1992) are used which have a strong 35S promoter and the CaMv polyadenylation signal.
The resulting plasmidε are tranεformed to A . tumefaciens strains. Strain EHA 105 (Hood, E.E. et al . , (1993) is used as a vector to transform tobacco basically as described by Rogers et al . (1986). Strain ClC58p-GV3850 (Zambryski et al . , (1983); Van Larabece et al . , (1974)) is used aε an alternative host to transform potato Solanum tuherosum (Desire) according to Dietze et al . (1995) .
The transformants are analyzed by Southern blot analysis to check for the presence of the genes. PCR-amplified, DIG- labelled (Boehringer) 200 bp gene fragments are used as a probe. The enzymatic activities of the cell extracts of transgenic plantε and the levels of εarcosine, dimethyl glycine and betaine are analyzed as described in Example 1.
Stress tolerance, for example, tolerance to drought, salinity, cold or freezing, resistance to pathogens, etc., is determined according to methods known in the art, for example, methods described in the technological background section of the present application. Example 13. Expression of E. halochloris GSMT and SDMT in rice
The plasmid constructionε described in Example 13 are also used to transfer the GSMT and SDMT genes to rice by particle bombardment. The GSMT are transferred to rice first and positive regenerated tranεformants are used as host plants for the SDMT transformation.
The following procedure are used. Immature Oryza sativa embryos of the Japonica variety Taipei 309 are aseptically isolated 10-14 days after pollination from greenhouse plants and plated scutulum site up on solid MS medium (Murashige and Skoog, 1962) containing 3% sucrose, 2 mg/1 2,4- dichlorophenoxyacetic acid and 50 mg/l cefotaxime (MSI) . After 4-6 days (28°C, darkness) embryos are transferred to εolid MS medium containing 10% εucroεe, 2 mg/1 2,4- dichlorophenoxyacetic acid and 50 mg/1 cefotaxime (MS2) and εubjected within 1 hour to microprojectile bombardment with a particle inflow gun. The DNA fragment containing the mehtyltransferase gene and the selective marker (5 μg) is precipitated on 1-3 mm gold particles (Aldrich) as described by Vain et al . , (1993) . Gold particles (400 mg per bombardment) are accelerated to the target with a particle inflow gun (Finer et al . , (1992) at a preεεure of 6 bar. Embryoε are placed 16 cm below the εyringe filter. Twenty four hourε poεt-bombardment embryos are subjected to selection on solid media (containing hygromycin or kanamycin) and incubated at 28°C in the dark.
After one week embryos are transferred to a liquid selection media, R2 medium (Ohira et al . , (1973) supplemented with: 3% sucrose, 1 mg/1 thiamine, 1 mg/1 2, 4-dichlorophenoxyacetic acid, 50 mg/1 cefotaxime and 20 mg/l hygromycin B or kanamycin. The embryos are incubated with shaking at 28°C in the dark and subcultured weekly. Developing calli are isolated 3 to 6 weeks later, and transferred to a callus increaεing media (R2 medium supplemented with: 6% εucrose, MS vitamins, 100 mg/l inositol, 2 mg/l 2, 4-dichlorophenoxyacetic acid, 50 mg/l cefotaxime and 20 mg/l hygromycin B kanamycin) . The calli are incubated in thiε media at 28°C in the dark and εubcultured weekly.
Reεiεtant calli are tranεferred to solid R2 regeneration media supplemented with 2% sucrose, 3% sorbitol, 20 mg/l hygromycin B, 1 mg/l zeatin, 0.5 mg/l indole-3-acetic acid, MS vitamins and 0.65% agarose. The callus tissue is maintained at 28°C with 12 h of light in order to enhance shoot formation. The calli are then subcultured every 3 weekε until εhootε had reached a length of 2-3 cm. They are transferred to half-strength MS rooting medium without hormones, supplemented with 1.5% sucroεe and 0.3% gelriteR (Sigma) . After 2-4 weeks of cultivation, plantlets are tranεferred directly to the green-house and planted in soil. Plantlets are grown in 7 liter aquaculture potε with fertilizer enriched earth, 3 plants per pot (day: 12 h, 28°C, 80% humidity; night: 12h, 21°X, 60% humidity) until they flower and set seeds.
To check the presence of the transgene, complexity of inεertion(ε) and number of copieε preεent, Southern blot analyεiε is performed as described previously (Burkhardt et al., 1997). A PCR amplified, DIG-labelled (Boehringer) 200-bp fragment of the coding region of the GSMT or SDMT genes is used as a probe .
The enzymatic activities of the cell extracts of transgenic plants and the levels of εarcoεine, dimethyl glycine and betaine are analyzed aε described in Example 1.
Stress tolerance, for example, tolerance to drought, salinity, cold or freezing, resistance to pathogens, etc., is determined according to methods known in the art, for example, methods described in the technological background section of the present application. Example 14. Expression of the E. halochloris GSMT and SDMT in yeast
pYX242 plasmid (R&D systems, USA) was used for expressing the GSMT and SDMT genes in Saccharomyces cerevisiae . The plasmid used (pYX242) is a E. coli -Saccharomyces cerevisiae εhuttle vector containing a bacterial origin of replication and ampicillin reεiεtance gene, a yeast {S. cerevisiae) origin of replication from 2μm DNA, and the yeast LEU2 gene for selection in yeast. The two genes are expressed under the yeast triose phosphate isomerase (TPI) promoter.
The DNA of the plaεmid pEFU described in Example 10 waε used as the template of PCR reactions. The primers uεed hybridize to DNA εequenceε shown in Fig. 8 (primer 1 and primer 4) and thus amplify both the GSMT and SDMT genes. The PCR fragment was ligated to the promoter of the expression plasmid with standard methods. A fragment containing a TPI transcription terminator and a fragment containing the TPI promoter was ligated between the two genes. Thus, both geneε are expressed under the TPI promoter. The primers used in the amplification of the fragments ligated in the expression plasmid contained suitable restriction sites that were used in the cloning. S. cerevisiae GRF18 (MATα, leu2-3, 11, his3- 11.15) was used as the hoεt for tranεformation. The transformation was performed according to Ito et al . uεing the εtandard lithium chloride procedure (Ito et al . , (1983) Bacteriol. 153, 163-170) using the LEU2 marker of pYX242 for selection.
The transformants were grown in YNB-medium supplemented with amino acid mixture without leucine (R &D systems product manual) . The cultivation was done overnight at 30°C by εhaking at 180 rpm. 5 ml of culture supernatant was centrifuged (1,700 g, 10 min) and the cell pellet waε suspended in 200 μl of the assay buffer (see example 1) supplemented with 1 mM PMSF. The cells were broken by vortexing with glass beads (10 x l min intervals) . The cells were kept on ice between the pulses. The cell debris was centrifuged down (30 min, 4°C, 10,000g).
The methylase activities were assayed from the supernatant as described in example 1. The relative enzyme activities on different substrates were the following: glycine - 17,000 dpm/30 min; sarcoεine - 71,000 dpm/30 min and dimethyl glycine - 530,000 dpm/30 min.
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Claims

What is claimed:
1. A nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID N0:1, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a) , (b) and (c) .
2. A nucleic acid molecule according to claim 1 which compriseε the DNA sequence from nucleotide 208 to 1047 of SEQ ID N0:1.
3. A nucleic acid molecule according to claim 1 which comprises the DNA sequence from nucleotide 221 to 1024 Of SEQ ID NO: 5.
4. A nucleic acid molecule according to claim 1 which compriεeε the DNA εequence from nucleotide 1048 to 1902 Of SEQ ID NO : 1.
5. A nucleic acid molecule according to claim 1 which compriεeε the DNA sequence from nucleotide 1031 to 1867 Of SEQ ID NO : 5.
6. A nucleic acid molecule according to claim 1 which comprises the DNA sequence from nucleotide 208 to 1902 of SEQ ID N0:1.
7. A nucleic acid molecule according to claim 1 which comprises the DNA sequence from nucleotide 221 to 1867 Of SEQ ID NO : 5.
8. A nucleic acid molecule according to claim 1 which comprises the DNA sequence from nucleotide 208 to 2722 of SEQ ID NO:l.
9. A nucleic acid molecule according to claim 1 which comprises the DNA sequence from nucleotide 221 to 3004 of SEQ ID NO : 5.
10. A nucleic acid molecule encoding a methyltransferase capable of catalyzing at least one of the reactions glycine to sarcosine, sarcoεine to dimethyl glycine and dimethyl glycine to betaine.
11. A nucleic acid molecule comprising a nucleotide εequence εelected form the group consisting of:
(a) a DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO:l, a DNA sequence from nucleotide 2006 to 3004 of SEQ ID N0:5,
(b) a nucleotide sequence which hybridizes to any of the sequenceε as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequenceε defined in (a) , (b) and (c) .
12. A nucleic acid molecule according to claim 11 which comprises the DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO:l.
13. A nucleic acid molecule according to claim 11 which comprises the DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO: 5.
14. A methyltransferaεe encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of :
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID N0:1, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID N0:5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID N0:1, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) ,
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequenceε defined in (a) , (b) and (c) .
15. A methyltransferase according to claim 14 encoded by a nucleic acid molecule having the DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l.
16. A methyltransferase according to claim 14 encoded by a nucleic acid molecule having the DNA sequence from nucleotide 221 to 1024 of SEQ ID N0:5.
17. A methyltransferase according to claim 14 encoded by a nucleic acid molecule having the DNA sequence from nucleotide 1048 to 1902 of SEQ ID N0:1.
18. A methyltransferase according to claim 14 encoded by a nucleic acid molecule having the DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO:5.
19. A methyltransferase comprising an amino acid sequence selected from the group consisting of:
(a) an amino acid sequence as depicted in SEQ ID NO:2, an amino acid sequence as depicted in SEQ ID NO:3, an amino acid sequence as depicted in SEQ ID NO: 6, an amino acid sequence as depicted in SEQ ID NO : 7 ,
(b) a fragment of an amino acid sequence as defined in (a) and
(c) a derivative of an amino acid sequence as defined in (a) and (b) .
20. A methyltransferase according to claim 19 having the amino acid sequence depicted in SEQ ID NO: 2.
21. A methyltransferase according to claim 19 having the amino acid sequence depicted in SEQ ID NO: 3.
22. A methyltransferaεe according to claim 19 having the amino acid sequence depicted in SEQ ID NO: 6.
23. A methyltransferase according to claim 19 having the amino acid εequence depicted in SEQ ID NO: 7.
24. A methyltransferase according to claim 19 having the amino acid sequence depicted in SEQ ID NO: 2 and SEQ ID NO: 3, wherein the N-terminus of SEQ ID NO: 3 is covalently joined to the C-terminus of SEQ ID NO: 2.
25. A methyltransferaεe capable of catalyzing at leaεt one of the reactionε glycine to εarcoεine, sarcosine to dimethyl glycine and dimethyl glycine to betaine.
26. A S-adenosyl methionine synthase encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
(a) a DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO:l, a DNA εequence from nucleotide 2006 to 3004 of SEQ ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) ,
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequenceε defined in (a) , (b) and (c) .
27. A S-adenosyl methionine synthase encoded by a nucleic acid molecule having the DNA εequence from nucleotide 2027 to 2722 of SEQ ID NO:l.
28. A S-adenosyl methionine synthase encoded by a nucleic acid molecule having the DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO: 5.
29. A S-adenosyl methionine synthase comprising an amino acid sequence selected form the group consisting of:
(a) an amino acid sequence as depicted in SEQ ID NO: 4 or an amino acid sequence as depicted in SEQ ID NO: 8,
(b) a fragment of an amino acid sequence as defined in (a) and
(c) a derivative of an amino acid sequence as defined in (a) and (b) .
30. A S-adenosyl methionine εynthaεe according to claim 26 having the amino acid sequence depicted in SEQ ID NO:4.
31. A S-adenoεyl methionine εynthaεe according to claim 26 having the amino acid εequence depicted in SEQ ID NO: 8.
32. An expression vector comprising at least one nucleotide sequence selected from the group conεiεting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO:5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequenceε aε defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequenceε defined in (a), (b)and (c) .
33. An expreεεion vector according to claim 32 compriεing the DNA εequence from nucleotide 208 to 1047 of SEQ ID NO : 1.
34. An expression vector according to claim 32 comprising the DNA sequence from nucleotide 221 to 1024 of SEQ ID NO : 5.
35. An expression vector according to claim 32 comprising the DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO : 1.
36. An expression vector according to claim 32 comprising the DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5.
37. An expression vector according to claim 32 comprising the DNA sequence from nucleotide 208 to 1902 of SEQ ID NO:l.
38. An expression vector according to claim 32 comprising the DNA sequence from nucleotide 221 to 1867 of SEQ ID NO: 5.
39. An expression vector comprising a nucleotide sequence coding for an enzyme capable of increaεing the amount of intracellular S-adenoεyl methionine and at leaεt one nucleotide sequence selected from the group consisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which iε degenerate aε a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) .
40. A recombinant organism transformed with at least one nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of :
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate aε a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) .
41. A recombinant organism according to claim 40 which is tranεformed with an expression vector comprising the DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l.
42. A recombinant organism according to claim 40 which is transformed with an expression vector comprising the DNA sequence from nucleotide 221 to 1024 of SEQ ID N0:5.
43. A recombinant organism according to claim 40 which is transformed with an expresεion vector comprising the DNA sequence from nucleotide 1048 to 1902 of SEQ ID N0:1.
44. A recombinant organism according to claim 40 which is transformed with an expresεion vector compriεing the DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5.
45. A recombinant organism according to claim 40 which is tranεformed with an expresεion vector compriεing the DNA sequence from nucleotide 208 to 1902 of SEQ ID NO:l.
46. A recombinant organiεm according to claim 40 which iε transformed with an expression vector compriεing the DNA εequence from nucleotide 221 to 1867 of SEQ ID NO:5.
47. A recombinant organism transformed with an nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increasing the amount of intracellular S-adenosyl methionine and at least one nucleotide sequence selected from the group consisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID N0:1, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID N0:1, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a reεult of the genetic code to any of the nucleotide εequences defined in (a), (b) and (c) .
48. A recombinant organism transformed with an nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increasing the amount of intracellular glycine and at least one nucleotide sequence selected from the group consisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO:5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequenceε defined in (a), (b)and (c) .
49. A recombinant organism according to claim 40, wherein the organism is a bacteria.
50. A recombinant organism according to claim 49, wherein the bacteria is selected from the group conεiεting of E. coli, Bacilluε, Corynebacteria, Pεeudomonas lactic acid bacteria and Streptomyces .
51. A recombinant organism according to claim 40, wherein the organism is a yeast.
52. A recombinant organism according to claim 51, wherein the yeast is selected from the group consisting of Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida and Hansenula.
53. A recombinant organism according to claim 40, wherein the organiεm iε a fungus .
54. A recombinant organism according to claim 53, wherein the fungus is selected from the group consisting of Aspergillus, Trichoderma and Penicillium.
55. A recombinant organism according to claim 40, wherein the organism is a plant selected from the group comprising cereals, legumeε, oilεeedε, vegetableε, fruits, ornamentals and perennial trees .
56. A recombinant organism according to claim 55, wherein the plant is selected from the group consisting of lettuces, Capsicumε, grasses, clovers, alfalfa, beans, sweet potatoes, cassava, yams, taro, groundnut, brassica, εugar beet, grapeε, potato, tomato, rice, tobacco, rapeseed, maize, sorghum, cotton, soybean, barley, wheat, rye, canola, sunflower, linseed, pea, cucumber, carrot, ornamentals, perennial trees and fruits.
57. A recombinant organism according to claim 47, wherein the organism is a bacteria.
58. A recombinant organism according to claim 57, wherein the bacteria is selected from the group consisting of E. coli, Bacillus, Corynebacteria, Pseudomonaε lactic acid bacteria and Streptomyces .
59. A recombinant organism according to claim 47, wherein the organiεm iε a yeaεt .
60. A recombinant organism according to claim 59, wherein the yeast is selected from the group consiεting of Saccharomyceε, Zygoεaccharomyces, Pichia, Kluyveromyces, Candida and Hansenula.
61. A recombinant organism according to claim 47, wherein the organism is a fungus .
62. A recombinant organism according to claim 61, wherein the fungus is selected from the group consiεting of Aspergillus, Trichoderma and Penicillium.
63. A recombinant organism according to claim 47, wherein the organism is a plant selected from the group comprising cereals, legumes, oilseeds, vegetables, fruits, ornamentals and perennial trees .
64. A recombinant organism according to claim 63, wherein the plant is selected from the group consisting of lettuces, Capsicumε, grasses, clovers, alfalfa, beans, sweet potatoes, cassava, yams, taro, groundnut, brasεica, εugar beet, grapeε, potato, tomato, rice, tobacco, rapeεeed, maize, εorghum, cotton, εoybean, barley, wheat, rye, canola, εunflower, linεeed, pea, cucumber, carrot, ornamentalε, perennial treeε and fruits.
65. A recombinant organism according to claim 48, wherein the organism iε a bacteria.
66. A recombinant organiεm according to claim 66, wherein the bacteria iε εelected from the group consisting of E. coli, Bacillus, Corynebacteria, Pseudomonas lactic acid bacteria and Streptomyces .
67. A recombinant organism according to claim 48, wherein the organism is a yeast .
68. A recombinant organiεm according to claim 67, wherein the yeaεt iε selected from the group consisting of Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida and Hansenula.
69. A recombinant organism according to claim 48, wherein the organism is a fungus .
70. A recombinant organism according to claim 69, wherein the fungus is selected from the group conεiεting of Aεpergillus, Trichoderma and Penicillium.
71. A recombinant organism according to claim 48, wherein the organiεm is a plant selected from the group comprising cereals, legumes, oilseeds, vegetables, fruits, ornamentals and perennial trees .
72. A recombinant organism according to claim 71, wherein the plant is εelected from the group consisting of lettuces, Capsicums, grasses, clovers, alfalfa, beans, sweet potatoes, cassava, yams, taro, groundnut, brasεica, εugar beet, grapeε, potato, tomato, rice, tobacco, rapeεeed, maize, εorghum, cotton, εoybean, barley, wheat, rye, canola, sunflower, linseed, pea, cucumber, carrot, ornamentals, perennial trees and fruits.
73. A method for the production of a recombinant organism comprising the steps of transforming a host organiεm with at least one nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of :
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide εequence as defined in (a) and (b) , and (d) a nucleotide sequence which iε degenerate as a result of the genetic code to any of the nucleotide sequenceε defined in (a), (b)and (c) .
74. A method for the production of a recombinant organism comprising the steps of transforming a host organism with a nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increasing the amount of intracellular S-adenosyl methionine and at least one a nucleotide sequence selected from the group consisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizeε to any of the εequences as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequenceε defined in (a), (b) and (c) .
75. A method for the production of a recombinant organism compriεing the steps of transforming a host organiεm with a nucleic acid molecule compriεing a nucleotide εequence coding for an enzyme capable of increaεing the amount of intracellular glycine and at least one a nucleotide εequence εelected from the group consisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide εequence as defined in (a) and (b) , and
(d) a nucleotide εequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c)
76. A methyltransferase obtainable by culturing an organism transformed with at leaεt one nucleic acid molecule comprising a nucleotide sequence εelected from the group consisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) and isolating the methyltransferase from said organism or the medium used to culture said organism.
77. A method for the production of a methyltransferase compriεing the steps of culturing an organism transformed with at least one nucleic acid molecule comprising a nucleotide εequence εelected from the group conεiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO:5,
(b) a nucleotide sequence which hybridizes to any of the εequenceε aε defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide εequences defined in (a), (b)and (c) , and iεolating the methyltransferase from said organism or the medium used to culture said organism.
78. A method for the production of sarcoεine comprising the steps of culturing an organiεm transformed with a nucleic acid molecule comprising a nucleotide εequence selected from the group consiεting of :
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and (d) a nucleotide εequence which is degenerate as a result of the genetic code to any of the nucleotide sequenceε defined in (a), (b)and (c) , and iεolating dimethyl glycine from εaid organiεm or the medium uεed to culture or proceεε εaid organism.
79. A method for the production of sarcosine comprising the steps of culturing an organiεm transformed with a nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increasing the amount of intracellular S-adenosyl methionine and at leaεt one a nucleotide εequence selected from the group consiεting of :
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO:5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizeε to any of the εequences as defined in (a) ,
(c) a fragment of a nucleotide sequence aε defined in (a) and (b) , and
(d) a nucleotide sequence which iε degenerate aε a result of the genetic code to any of the nucleotide εequenceε defined in (a), (b)and (c) , and iεolating εarcosine from said organism or the medium used to culture or process said organism.
80. A method for the production of εarcosine comprising the steps of culturing an organism transformed with a nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increasing the amount of intracellular glycine and at least one a nucleotide εequence εelected from the group conεisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) , and isolating sarcoεine from said organism or the medium used to culture or procesε εaid organiεm.
81. A method for the production of dimethyl glycine compriεing the εteps of culturing an organism transformed with a nucleic acid molecule compriεing a nucleotide εequence εelected from the group consiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ ID NO:5,
(b) a nucleotide sequence which hybridizes to any of the εequenceε aε defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) , and isolating dimethyl glycine from εaid organism or the medium used to culture or proceεε said organism.
82. A method for the production of dimethyl glycine comprising the steps of culturing an organism transformed with a nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increaεing the amount of intracellular S- adenosyl methionine and at least one a nucleotide sequence selected from the group consiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO:5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizeε to any of the sequenceε as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide εequence which is degenerate as a result of the genetic code to any of the nucleotide εequenceε defined in (a), (b)and (c) , and isolating dimethyl glycine from said organism or the medium used to culture or process said organiεm.
83. A method for the production of dimethyl glycine comprising the steps of culturing an organism tranεformed with a nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increasing the amount of intracellular glycine and at leaεt one a nucleotide sequence selected from the group consisting of: (a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO:5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID N0:1, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide εequence as defined in (a) and (b) , and
(d) a nucleotide εequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) , and iεolating dimethyl glycine from εaid organism or the medium used to culture or procesε εaid organiεm.
84. A method for the production of betaine, compriεing the εtepε of culturing an organiεm tranεformed with at leaεt one nucleic acid molecule comprising a nucleotide εequence selected from the group consisting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and (d) a nucleotide εequence which is degenerate as a reεult of the genetic code to any of the nucleotide εequences defined in (a), (b)and (c) , and isolating betaine from εaid organiεm or the medium uεed to culture or process said organism.
85. A method for the production of betaine, comprising the steps of culturing an organism transformed with a nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increasing the amount of intracellular S-adenosyl methionine and at leaεt one nucleotide εequence εelected from the group conεiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID N0:1, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID N0:1, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID N0:5,
(b) a nucleotide sequence which hybridizeε to any of the εequenceε as defined in (a) ,
(c) a fragment of a nucleotide sequence aε defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequenceε defined in (a), (b) and (c) , and iεolating betaine from εaid organism or the medium uεed to culture or proceεs said organism.
86. A method for the production of betaine, comprising the steps of culturing an organism tranεformed with a nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increasing the amount of intracellular glycine and at least one nucleotide sequence selected from the group consiεting of: (a) a DNA εequence from nucleotide 208 to 1047 of SEQ
ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO:5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the εequenceε as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide εequences defined in (a), (b)and (c) , and iεolating betaine from εaid organism or the medium used to culture or proceεs said organism.
87. A method for increasing the intracellular concentration of sarcoεine, dimethyl glycine or betaine in an organiεm compriεing the steps of transforming an organism with at leaεt one nucleic acid molecule compriεing a nucleotide sequence selected from the group consiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide εequence as defined in (a) and (b) , and
(d) a nucleotide sequence which iε degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) , such that the nucleic acid molecule (s) is expressed.
88. A method for increasing the intracellular concentration of εarcoεine, dimethyl glycine or betaine in an organism comprising the steps of transforming an organism with a nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increaεing the amount of intracellular S-adenoεyl methionine and at leaεt one nucleotide sequence selected from the group conεiεting of:
(a) a DNA sequence from nucleotide 208 to11047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) .
89. A method for increasing the intracellular concentration of sarcosine, dimethyl glycine or betaine in an organism comprising the stepε of tranεforming an organiεm with a nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increasing the amount of intracellular glycine and at least one nucleotide εequence εelected from the group consisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID N0:1, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizeε to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide εequence which is degenerate as a result of the genetic code to any of the nucleotide εequenceε defined in (a), (b)and (c) .
90. A method for enhancing the salt tolerance of an organism comprising the εteps of transforming an organism with at least one nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide εequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a reεult of the genetic code to any of the nucleotide εequenceε defined in (a), (b)and (c) , εuch that the nucleic acid molecule (ε) is expressed.
91. A method for enhancing the salt tolerance of an organism comprising the stepε of transforming an organiεm with a nucleic acid molecule compriεing a nucleotide sequence coding for an enzyme capable of increaεing the amount of intracellular S-adenoεyl methionine and at least one nucleic acid molecule comprising a nucleotide sequence selected from the group consiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide εequence as defined in
(a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) .
92. A method for enhancing the salt tolerance of an organiεm comprising the stepε of tranεforming an organiεm with a nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increasing the amount of intracellular glycine and at least one nucleic acid molecule compriεing a nucleotide εequence εelected from the group conεiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ ID N0:5, a DNA sequence from nucleotide 1048 to 1902 of SEQ ID N0:1, a DNA εequence from nucleotide 1031 to 1867 of SEQ ID NO: 5, (b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence aε defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide εequences defined in (a), (b)and (c) .
93. A method for enhancing the freezing or cold tolerance of an organism comprising the steps of transforming an organism with at least one nucleic acid molecule comprising a nucleotide sequence selected from the group conεisting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO:5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO:5,
(b) a nucleotide sequence which hybridizes to any of the εequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide εequences defined in (a), (b)and (c) , such that the nucleic acid molecule (ε) iε expreεsed.
94. A method for enhancing the freezing or cold tolerance of an organiεm compriεing the εtepε tranεforming an organiεm with a nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increaεing the amount of intracellular S-adenoεyl methionine and at leaεt one nucleic acid molecule comprising a nucleotide εequence selected from the group conεiεting of: (a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) .
95. A method for enhancing the freezing or cold tolerance of an organiεm compriεing the εtepε tranεforming an organiεm with a nucleic acid molecule compriεing a nucleotide εequence coding for an enzyme capable of increaεing the amount of intracellular glycine and at leaεt one nucleic acid molecule comprising a nucleotide εequence selected from the group consisting of :
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID N0:1, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO.l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO:5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide εequence as defined in (a) and (b) , and (d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a), (b) and (c) .
96. A method for enhancing the resistance of an organiεm to drought or water stress comprising the steps of transforming an organiεm with at least one nucleic acid molecule compriεing a nucleotide sequence εelected from the group conεiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID N0:1, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequenceε aε defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide εequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) , such that the nucleic acid molecule (ε) iε expreεεed.
97. A method for enhancing the reεiεtance of an organiεm to drought or water streεε compriεing the εteps of transforming an organiεm with a nucleic acid molecule comprising a nucleotide εequence coding for an enzyme capable of increasing the amount of intracellular S-adenosyl methionine and at least one nucleic acid molecule comprising a nucleotide sequence selected from the group conεiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO:5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO:5,
(b) a nucleotide εequence which hybridizeε to any of the εequences as defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate aε a reεult of the genetic code to any of the nucleotide sequences defined in (a), (b)and' (c) .
98. A method for enhancing the resiεtance of an organiεm to drought or water stress comprising the steps of transforming an organiεm with a nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increaεing the amount of intracellular glycine and at leaεt one nucleic acid molecule comprising a nucleotide sequence εelected from the group consiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the εequenceε as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate aε a reεult of the genetic code to any of the nucleotide εequences defined in (a), (b)and (c) .
99. A method for enhancing the productivity or yield of an organiεm compriεing the εteps of transforming an organiεm with at leaεt one nucleic acid molecule compriεing a nucleotide εequence selected from the group conεiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizes to any of the sequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate aε a reεult of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) , such that the nucleic acid molecule (ε) is expreεεed.
100. A method for enhancing the productivity or yield of an organiεm compriεing the εteps of transforming an organism with a nucleic acid molecule comprising a nucleotide εequence coding for an enzyme capable of increaεing the amount of intracellular S-adenosyl methionine and at leaεt one nucleic acid molecule compriεing a nucleotide εequence εelected from the group conεiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID N0:1, a DNA εequence from nucleotide 221 to 1024 of SEQ ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ ID NO: 5, (b) a nucleotide εequence which hybridizeε to any of the εequenceε as defined in (a) ,
(c) a fragment of a nucleotide εequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide εequenceε defined in (a), (b) and (c) .
101. A method for enhancing the productivity or yield of an organiεm compriεing the εtepε of tranεforming an organiεm with a nucleic acid molecule compriεing a nucleotide εequence coding for an enzyme capable of increaεing the amount of intracellular glycine and at leaεt one nucleic acid molecule compriεing a nucleotide εequence εelected from the group conεiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizeε to any of the εequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence as defined in (a) and (b) , and
(d) a nucleotide sequence which iε degenerate aε a reεult of the genetic code to any of the nucleotide εequenceε defined in (a), (b)and (c) .
102. A method for inducing pathogeneεiε-related proteinε in a plant compriεing the εtepε of tranεforming a plant with at leaεt one nucleic acid molecule compriεing a nucleotide sequence selected from the group conεiεting of: (a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence aε defined in (a) and (b) , and
(d) a nucleotide εequence which is degenerate as a result of the genetic code to any of the nucleotide sequenceε defined in (a), (b)and (c) , εuch that the nucleic acid molecule (ε) iε expreεεed.
103. A method for inducing pathogeneεiε-related proteinε in a plant compriεing the εtepε of transforming a plant with a nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increaεing the amount of intracellular S-adenoεyl methionine and at leaεt one nucleic acid molecule compriεing a nucleotide εequence selected from the group consisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequenceε aε defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and (d) a nucleotide sequence which is degenerate aε a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) .
104. A method for inducing pathogenesis-related proteins in a plant compriεing the εteps of tranεforming a plant with a nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increasing the amount of intracellular glycine and at least one nucleic acid molecule comprising a nucleotide εequence selected from the group consisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence aε defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate aε a reεult of the genetic code to any of the nucleotide εequences defined in (a), (b)and (c) .
105. A method for increasing the resistance of a plant to attack by pathogens comprising the stepε of transforming a plant with at least one nucleic acid molecule comprising a nucleotide sequence selected from the group consiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID N0:1, a DNA εequence from nucleotide 221 to 1024 of SEQ ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the εequenceε as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide εequences defined in (a), (b)and (c) , such that the nucleic acid molecule (s) is expresεed.
106. A method for increaεing the reεiεtance of a plant to attack by pathogenε comprising the steps of transforming a plant with a nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increaεing the amount of intracellular S-adenoεyl methionine and at leaεt one nucleic acid molecule compriεing a nucleotide εequence εelected from the group conεiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID N0:1, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID N0:1, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequenceε aε defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide εequenceε defined in (a), (b)and (c) .
107. A method for increasing the resistance of a plant to attack by pathogenε compriεing the steps of tranεforming a plant with a nucleic acid molecule comprising a nucleotide εequence coding for an enzyme capable of increaεing the amount of intracellular glycine and at leaεt one nucleic acid molecule compriεing a nucleotide εequence εelected from the group consisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO:5,
(b) a nucleotide sequence which hybridizes to any of the εequenceε as defined in (a) ,
(c) a fragment of a nucleotide εequence as defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate aε a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) .
108. A method for improving the nutritional value of a plant comprising the εteps of tranεforming a plant with at leaεt one nucleic acid molecule comprising a nucleotide sequence εelected from the group conεisting of: (a) a DNA sequence from nucleotide 208 to 1047 of SEQ
ID N0:1, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5, (b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequenceε defined in (a), (b)and (c) , εuch that the nucleic acid molecule (s) is expreεsed.
109. A method for improving the nutritional value of a plant comprising the stepε of tranεforming a plant with a nucleic acid molecule compriεing a nucleotide εequence coding for an enzyme capable of increaεing the amount of intracellular S-adenoεyl methionine and at leaεt one nucleic acid molecule compriεing a nucleotide sequence selected from the group consiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the εequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate aε a reεult of the genetic code to any of the nucleotide εequenceε defined in (a), (b)and (c) .
110. A method for improving the nutritional value of a plant compriεing the εteps of tranεforming a plant with a nucleic acid molecule compriεing a nucleotide sequence coding for an enzyme capable of increasing the amount of intracellular glycine and at leaεt one nucleic acid molecule compriεing a nucleotide εequence εelected from the group consisting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID N0:5,
(b) a nucleotide εequence which hybridizes to any of the sequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate aε a reεult of the genetic code to any of the nucleotide εequences defined in (a), (b)and (c) .
Ill . A method for enhancing the pH tolerance of a cultured microorganiεm compriεing the εtepε of tranεforming a microorganiεm with at leaεt one nucleic acid molecule comprising a nucleotide sequence εelected from the group conεiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizeε to any of the εequences as defined in (a) ,
(c) a fragment of a nucleotide εequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide εequenceε defined in (a), (b)and (c) , εuch that the nucleic acid molecule (ε) iε expreεsed.
112. A method for enhancing the pH tolerance of a cultured microorganism comprising the stepε of transforming a microorganiεm with a nucleic acid molecule compriεing a nucleotide εequence coding for an enzyme capable of increaεing the amount of intracellular S-adenoεyl methionine and at least one nucleic acid molecule comprising a nucleotide εequence εelected from the group conεisting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:'l, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizes to any of the εequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequenceε defined in (a), (b)and (c) .
113. A method for enhancing the pH tolerance of a cultured microorganiεm comprising the steps of tranεforming a microorganiεm with a nucleic acid molecule compriεing a nucleotide sequence coding for an enzyme capable of increasing the amount of intracellular glycine and at least one nucleic acid molecule comprising a nucleotide sequence εelected from the group conεisting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence as defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a), (b) and (c) .
114. A method for improving the viability of a cultured microorganism comprising the stepε of tranεforming an microorganiεm with at leaεt one nucleic acid molecule compriεing a nucleotide εequence selected from the group consiεting of :
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide εequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequenceε defined in (a), (b)and (c) , εuch that the nucleic acid molecule (ε) iε expresεed.
115. A method for improving the viability of a cultured microorganism comprising the steps of transforming a microorganism with a nucleic acid molecule comprising a nucleotide εequence coding tor an enzyme capable of increaεing the amount of intracellular S-adenoεyl methionine and at leaεt one nucleic acid molecule compriεing a nucleotide εequence εelected from the group conεiεting of :
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide sequence which iε degenerate aε a reεult of the genetic code to any of the nucleotide εequences defined in (a), (b)and (c) .
116. A method for improving the viability of a cultured microorganiεm comprising the εtepε of tranεforming a microorganism with a nucleic acid molecule comprising a nucleotide εequence coding for an enzyme capable of increaεing the amount of intracellular glycine and at leaεt one nucleic acid molecule compriεing a nucleotide sequence εelected from the group consiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ ID N0:1, a DNA εequence from nucleotide 1031 to 1867 of SEQ ID NO: 5, (b) a nucleotide sequence which hybridizes to any of the sequenceε aε defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequenceε defined in (a), (b)and (c) .
117. A method for decreaεing inclusion body formation in a microorganism expresεing a heterologouε protein compriεing the εtepε of tranεforming a microorganiεm with a nucleic acid molecule capable of expreεεing a heterologouε protein and tranεforming said microorganiεm with at leaεt one nucleic acid molecule compriεing a nucleotide sequence selected from the group consiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizes to any of the sequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate aε a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) , εuch that the nucleic acid oleculeε are expreεεed.
118. A method for decreaεing incluεion body formation in a microorganism expressing a heterologous protein comprising the εtepε of tranεforming a microorganiεm with a nucleic acid molecule capable of expreεεing a heterologouε protein and tranεforming εaid microorganiεm with a nucleic acid molecule compriεing a nucleotide εequence coding for an enzyme capable of increasing the amount of intracellular S-adenoεyl methionine and at least one nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of :
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO:5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO:5,
(b) a nucleotide sequence which hybridizes to any of the sequenceε as defined in (a) ,
(c) a fragment of a nucleotide sequence aε defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate as a result of the genetic code to any of the nucleotide sequenceε defined in (a), (b)and (c) .
119. A method for decreaεing incluεion body formation in a microorganiεm expreεεing a heterologous protein comprising the steps of tranεforming a microorganiεm with a nucleic acid molecule capable of expreεεing a heterologouε protein and tranεforming εaid microorganiεm with a nucleic acid molecule compriεing a nucleotide εequence coding for an enzyme capable of increaεing the amount of intracellular glycine and at leaεt one nucleic acid molecule compriεing a nucleotide εequence εelected from the group conεiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ ID NO: 5,
(b) a nucleotide εequence which hybridizes to any of the sequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide εequences defined in (a), (b)and (c) .
120. A method for increasing the stability of a heterologous protein expresεed in a microorganiεm compriεing the steps of transforming a microorganiεm with a nucleic acid molecule capable of expreεsing a heterologous protein and transforming said microorganiεm with at leaεt one nucleic acid molecule compriεing a nucleotide εequence εelected from the group conεiεting of:
(a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID N0:1, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence as defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide εequenceε defined in (a), (b)and (c) , εuch that the nucleic acid molecules are expressed.
121. A method for increaεing the εtability of a heterologouε protein expreεεed in a microorganiεm compriεing the εteps of tranεforming a microorganiεm with a nucleic acid molecule capable of expreεεing a heterologouε protein and transforming said microorganism with a nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increasing the amount of intracellular S-adenosyl methionine and at least one nucleic acid molecule comprising a nucleotide sequence selected from the group consiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a reεult of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) .
122. A method for increasing the stability of a heterologous protein expreεεed in a microorganiεm compriεing the εtepε of tranεforming a microorganiεm with a nucleic acid molecule capable of expresεing a heterologouε protein and tranεforming said microorganiεm with a nucleic acid molecule compriεing a nucleotide εequence coding for an enzyme capable of increaεing the amount of intracellular glycine and at leaεt one nucleic acid molecule compriεing a nucleotide εequence εelected from the group conεiεting of: (a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID N0:1, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizeε to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate as a result of the genetic code to any of the nucleotide sequenceε defined in (a), (b)and (c) .
123. A method for increaεing the production of a heterologouε protein expreεεed in a microorganiεm comprising the stepε of tranεforming a microorganism with a nucleic acid molecule capable of expreεεing a heterologous protein and transforming said microorganism with at leaεt one nucleic acid molecule compriεing a nucleotide εequence εelected from the group conεiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizeε to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence aε defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate aε a reεult of the genetic code to any of the nucleotide εequenceε defined in (a), (b)and (c) , εuch that the nucleic acid moleculeε are expreεεed.
124. A method for increasing the production of a heterologous protein expresεed in a microorganiεm compriεing the εtepε of tranεforming a microorganiεm with a nucleic acid molecule capable of expreεεing a heterologouε protein and tranεforming εaid microorganiεm with a nucleic acid molecule compriεing a nucleotide εequence coding for an enzyme capable of increaεing the amount of intracellular S-adenoεyl methionine and at leaεt one nucleotide εequence selected from the group consisting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizeε to any of the εequences aε defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate aε a reεult of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) .
125. A method for increasing the production of a heterologouε protein expreεεed in a microorganiεm compriεing the εtepε of transforming a microorganism with a nucleic acid molecule capable of expreεεing a heterologouε protein and tranεforming εaid microorganiεm with a nucleic acid molecule compriεing a nucleotide εequence coding for an enzyme capable of increaεing the amount of intracellular glycine and at least one nucleotide sequence selected from the group consiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID N0:1, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID N0:1, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizes to any of the sequences as defined in (a) ,
(c) a fragment of a nucleotide sequence as defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate as a result of the genetic code to any of the nucleotide sequenceε defined in (a), (b)and (c) .
126. An animal feed compriεing a recombinant organiεm transformed with at least one nucleic acid molecule compriεing a nucleotide εequence εelected from the group conεiεting of :
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizeε to any of the εequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide εequence which is degenerate as a result of the genetic code to any of the nucleotide εequenceε defined in (a), (b)and (c) .
127. An animal feed comprising a recombinant organism tranεformed with a nucleic acid molecule comprising a nucleotide sequence coding for an enzyme capable of increasing the amount of intracellular S- adenoεyl methionine and at least one nucleotide sequence εelected from the group conεiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizes to any of the sequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) .
128. An animal feed comprising a recombinant organiεm tranεformed with a nucleic acid molecule compriεing a nucleotide εequence coding for an enzyme capable of increaεing the amount of intracellular glycine and at leaεt one nucleotide εequence εelected from the group conεiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID N0:1, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID N0:5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID N0:1, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the sequences as defined in (a) , (c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate aε a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) .
129. An animal feed ingredient comprising a recombinant organism transformed with at least one nucleic acid molecule comprising a nucleotide εequence εelected from the group conεiεting of :
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID N0:1, a DNA sequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide εequence which hybridizes to any of the sequenceε as defined in (a) ,
(c) a fragment of a nucleotide εequence as defined in (a) and (b) , and
(d) a nucleotide εequence which iε degenerate aε a reεult of the genetic code to any of the nucleotide εequenceε defined in (a), (b)and (c) .
130. An animal feed ingredient comprising a recombinant organiεm tranεformed with a nucleic acid molecule compriεing a nucleotide εequence coding for an enzyme capable of increaεing the amount of intracellular S-adenoεyl methionine and at leaεt nucleotide εequence εelected from the group conεiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID N0:1, a DNA εequence from nucleotide 221 to 1024 of SEQ ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ ID NO: 5,
(b) a nucleotide εequence which hybridizes to any of the sequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide εequence which is degenerate as a reεult of the genetic code to any of the nucleotide εequenceε defined in (a), (b) and (c) .
131. An animal feed ingredient compriεing a recombinant organism transformed with a nucleic acid molecule compriεing a nucleotide εequence coding for an enzyme capable of increaεing the amount of intracellular S-adenoεyl methionine and at least nucleotide sequence selected from the group conεiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ
ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ
ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ
ID NO: 5,
(b) a nucleotide sequence which hybridizes to any of the εequenceε aε defined in (a) ,
(c) a fragment of a nucleotide εequence aε defined in (a) and (b) , and
(d) a nucleotide εequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a), (b)and (c) .
132. A DNA probe for use in identifying and cloning a nucleic acid molecule encoding a methyltransferase compriεing at leaεt 15 nucleotideε of a nucleotide sequence selected from the group conεiεting of: (a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID N0:1, a DNA εequence from nucleotide 221 to 1024 of SEQ ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5.
133. A method for identifying and cloning a nucleic acid molecule encoding a methyltransferase comprising the εtepε of hybridizing a probe conεiεting of at leaεt 15 nucleotideε of a nucleotide εequence εelected from the group conεiεting of:
(a) a DNA εequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA εequence from nucleotide 221 to 1024 of SEQ ID NO: 5, a DNA εequence from nucleotide 1048 to 1902 of SEQ ID NO:l, a DNA εequence from nucleotide 1031 to 1867 of SEQ ID NO: 5, with a εample containing nucleic acid of an organiεm, detecting a nucleic acid molecule in εaid εample which hybridizes to εaid probe and isolating said detected nucleic acid molecule.
134. A method for the purification of a methyl- tranεferaεe capable of catalyzing the converεion of glycine to dimethyl glycine compriεing the εteps of subjecting a εample comprising the methyltransferaεe to a matrix containing adenoεine, binding εaid methyltransferaεe to εaid matrix and eluting εaid methyltranεferaεe from εaid matrix.
EP99944425A 1998-08-20 1999-08-18 Methyltransferases, nucleic acid molecules encoding methyltransferases, their recombinant expression and uses thereof Withdrawn EP1112352A2 (en)

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FR2698879B1 (en) * 1992-12-09 1995-01-13 Rhone Poulenc Biochimie capsules modified in the catabolism of betaine, preparation and uses, in particular for the production of metabolites or enzymes.
JP3149319B2 (en) * 1994-07-22 2001-03-26 オリエンタル酵母工業株式会社 Method for producing recombinant GMT
SE9604532D0 (en) * 1996-12-09 1996-12-09 Leif Buelow Transgenic plants having increased freezing tolerance

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